Pharmaco-molecular assessment of the effects of anandamide and its antagonists on hippocampal tissue in Wistar albino rats.

OBJECTIVE: The members of the matrix metalloproteinase (MMP) family and cannabinoids (CBs) are reportedly associated with hippocampus-dependent memory functions. However, the effects of endogenously formed CBs on hippocampal long-term potentiation remain unknown. The present study aimed to investigate the changes in the gene and protein expression levels of matrix metallopeptidase 9 (MMP-9), phosphatase and tensin homolog (PTEN), and NOTCH receptor 1 (NOTCH1) in rat hippocampal tissues treated with anandamide (AEA), AM251, 6-iodopravadolin (AM630), and N-[4-{[(3,4-Dimethyl-5-isoxazolyl)amino]sulfonyl}phenyl] (ML193). MATERIALS AND METHODS: The subjects were divided into 10 groups (n = five per group). The pharmaceuticals were administered via intraperitoneal injection once a day for seven days, except for the control group. The resected hippocampal tissues were then evaluated using a quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analysis. The data obtained were statistically analyzed, and p < 0.01 was considered statistically significant. RESULTS: Contrary to the literature, the changes in MMP-9 expression were not statistically significant, but the changes in PTEN and NOTCH1 were. The findings of this in vivo experimental study revealed that the agonists and antagonists acting on the CB system have significant molecular effects on hippocampal tissue. CONCLUSIONS: The changes in gene and protein expressions may be one of the reasons for the neurodegenerative processes observed in patients using these agonists and antagonists, whose effects on the CB system have not been fully explained yet. Our study can contribute to the literature as it is the first study investigating the MMP-9, PTEN and NOTCH1 gene and protein expression.

Infection of Mycobacterium tuberculosis Promotes Both M1/M2 Polarization and MMP Production in Cigarette Smoke-Exposed Macrophages.

Pulmonary tuberculosis (PTB) is a risk factor for COPD. Our previous study revealed more severe emphysema in COPD patients (mostly smokers) with prior tuberculosis. However, the mechanisms of interactions between cigarette smoke (CS) and Mycobacterium tuberculosis (Mtb) are unknown. In this study, we found that the frequencies of both M1 and M2 macrophages, and levels of MMP9 and MMP12 in bronchoalveolar lavage were increased in PTB patients with smoking. Between-group analysis showed that the frequency of M1 macrophages was higher in non-smoker PTB patients while more M2 macrophages were found in smokers without PTB, as compared to the non-smoker healthy controls. Bacille Calmette-Guérin (BCG) infection in CS extract (CSE)-incubated MH-S cells further enhanced secretion of M1-related (iNOS, IFN-γ and TNF-α) and M2-related (TGF-ß and IL-10) cytokines, reactive oxygen species (ROS) production and cellular apoptosis, concomitantly with up-regulation of MMP9 and MMP12, but not TIMP1. Moreover, BCG infection in acutely CS-exposed mice promoted macrophage polarization toward both M1 and M2 phenotypes, along with increased lung inflammatory infiltration. MMP9 and MMP12, but not TIMP1, were further up-regulated in lung tissues and BAL fluid after BCG infection in this model. Taken together, Mtb Infection promoted CS-exposed macrophages to polarize toward both M1 and M2 phenotypes, along with enhanced production of MMP9 and MMP12. These findings provide insights into the mechanistic interplay between CS exposure and tuberculosis in the pathogenesis of COPD.

Assessment of CMKLR1 level in colorectal cancer and its correlation with angiogenic markers.

INTRODUCTION: Colorectal cancer (CRC) is the second most common malignant neoplasm in men and third in women. It is also the third leading cause of cancer-related death, killing annually >700,000 patients in the world. The global burden of CRC is expected to increase by 60% to >2.2 million new cases and 1.1 million deaths by 2030. The pathogenesis of cancer mainly depends on angiogenesis. This process plays a key role in the growth and infiltration of tumors which is essential for distant metastases. A large number of biochemical pathways is involved in the regulation of angiogenesis. As a subject of our study, we chose chemerin/chemokine-like receptor 1 (CMKLR1) pathway which is responsible for the angiogenic processes in malignant neoplasms. AIM OF THE STUDY: To assess the CMKLR1 level and the concentrations of the two markers of angiogenesis, matrix metalloproteinase (MMP)-9 and vascular cell adhesion molecule (VCAM)-1, in tumor and margin tissues of CRC in relation to histological grade and TNM classification. MATERIALS AND METHODS: The study used 47 samples of tumor and margin tissues derived from CRC patients. To determine the concentration of CMKLR1, MMP-9, and VCAM-1, we used the commercially available enzyme-linked immunosorbent assay kit. RESULTS: We found a significantly higher concentration of CMKLR1 and MMP-9 in tumor tissue compared to margin. There was no difference in VCAM-1 concentration between tumor and margin. The margin concentration of CMKLR1 was significantly correlated with that of both MMP-9 and VCAM-1. The margin concentration of VCAM-1 was correlated with that of MMP-9. Additionally, we observed that the tumor levels of CMKLR1 and MMP-9 were positively correlated with the tumor size (T parameter). CONCLUSION: CMKLR1 activity may be associated with the angiogenic process in CRC via MMP-9 activity. Further research, involving a larger sample, may verify whether chemerin/CMKLR1 axis could be considered as a suitable target in novel molecular therapies.

Immunohistochemical assessment of metalloproteinases MMP2 and MMP9 expression in canine various subtypes of lymphomas in relation with proliferative and apoptotic markers.

Matrix metalloproteinases 2 and 9 (MMP2 and MMP9) are proteolytic enzymes involved with extracellular matrix degradation. They play a role in tumor invasion and metastases. Because of their ability to degrade signaling molecules presented in extracellular matrix, MMPs contribute to tumor proliferation and apoptosis. The aim of this study was to evaluate expression of MMP2 (latent and both active and latent forms) and MMP9 (active, latent, active and latent forms) in different subtypes of canine lymphomas and their relationship with proliferative (mitotic index and percentage of Ki67-positive cells) and apoptotic (apoptotic index) markers. Expression of MMPs was assessed immunohistochemically using an immunoreactive score system. Expression of both MMPs was found in all 20 examined lymphomas belonging to six subtypes. Most cases showed a moderate level of all analyzed forms of MMP2 and MMP9. High expression of MMPs was found in single cases. Except for a positive correlation between the active form of MMP9 and the mitotic index for all lymphoma cases, no other correlations between any remaining forms of MMPs and neither proliferative nor apoptotic markers were found, irrespective of whether the analysis encompassed all cases or the most numerous lymphoma subtypes i.e. centroblastic and Burkitt-like. Our results were not able to clearly confirm the influence of MMPs on the proliferation and apoptotic activity of canine lymphoma cells. However, further studies examining MMPs activity by zymography, expression of their inhibitors and other factors involved in activation of cell proliferation and apoptosis inhibition are needed to clarify the role of MMPs, especially the active form of MMP9, in the behavior of canine lymphoma cells.

Short communication: Assessment of biomarkers of inflammation in the vaginal discharge of postpartum dairy cows diagnosed with clinical metritis.

Clinical metritis, characterized by the presence of an enlarged uterus and abnormal red-brownish foul-smelling vaginal discharge (VD), is a prevalent condition that causes important economic losses to dairy operations. The accurate diagnosis and treatment of this disease can help decrease its negative effects on the well-being and performance of dairy cows. The objectives of this study were to assess (1) the concentration of haptoglobin (Hp) and neutrophil-derived haptoglobin-matrix metalloproteinase 9 (Hp-MMP 9) in the VD of postpartum cows; and (2) the correlation between Hp and Hp-MMP 9 concentrations in serum and VD. Fifty-three dairy cows from 4 farms in central Pennsylvania were enrolled in this observational study.. Postpartum cows (7 ± 3 DIM) were screened using a Metricheck device to assess VD score (VDS): 1, clear fluid (n = 4); 2, < 50% white purulent fluid (n = 14); 3, > 50% white purulent fluid (n = 8); 4, red-brownish watery fluid without fetid smell (n = 22); and 5, fetid red-brownish watery fluid (n = 5). Blood and VD samples were collected for assessment of Hp and Hp-MMP 9 concentrations. Cows with a VDS of 4 or 5 (VDS4/5) had higher serum Hp concentrations than cows with a VDS of 1, 2, or 3 (VDS1/3; 93 ± 187 µg/mL vs. 59 ± 106 µg/mL, respectively). Similarly, cows with VDS4/5 had higher VD Hp concentrations than cows with VDS1/3 (73 ± 56 µg/mL vs. 17 ± 16 µg/mL, respectively). We found a significant correlation (0.37) between Hp levels in serum and in VD. We found no difference in serum Hp-MMP 9 between VDS4/5 and VDS1/3 cows. The VD concentrations of Hp-MMP 9 were higher in VDS4/5 cows than in VDS1/3 cows (7,629 ± 9,847 ng/mL vs. 1,567 ± 2,165 ng/mL, respectively). The correlation between Hp-MMP 9 in serum and VD was 0.22; nevertheless, it was not statistically significant. Interestingly, Hp and Hp-MMP 9 concentrations were higher in VD samples than in serum, regardless of VDS. Results from this study suggest that inflammatory biomarkers may be increased in cows with a VDS of 4 or 5. Further research should be aimed at elucidating the processes involved in inflammatory biomarker production and transportation in the uterus, as well as the effect of these biomarkers on endometrial cells.

Assessment of the Inhibitory Effects of Ficin-hydrolyzed Gelatin Derived from Squid (Uroteuthis duvauceli) on Breast Cancer Cell Lines and Animal Model.

Marine novel natural products have been applied for cancer therapy. Enzyme-digested gelatin hydrolysates have proven to serve as promising sources of potent biologically active peptides. Potential anti-breast cancer properties of the extracted Ficin-digesterd gelatin hydrolysate from Indian squid (Uroteuthis duvauceli) was extensively characterized by cellular and animal models. Gelatin was extracted from squid skin, hydrolyzed by Ficin, and characterized by standard physico-chemical methods. Ficin-digested gelatin hydrolysate was used at various doses of 0-0.1 mg/mL for assessment of MCF-7 and MDA-MB-231 breast cancer cells versus HUVEC normal cells. Cytotoxicity, phase-contrast morphological examination, apoptosis/necrosis, clonal-growth, cell-migration, Matrix-metalloproteinases (MMPs) zymography, and Western blotting were used for cellular assessments. For animal studies, breast tumor-induced BALB/c mice received hydrolyzed gelatin regimen, followed by tumor size/growth and immune-histochemical analyses. Significant inhibition of MCF-7 and MDA-MB-231 with no cytotoxicity on HUVEC cells were detected. Apoptosis was increased in cancer cells, as revealed by elevated ratio of cleaved caspase-3 and PARP. MMP-2 and MMP-9 activities in both cancer cells were diminished. In mice, gelatin hydrolysate prevented weight loss, decreased tumor size, induced p53, and down-regulated Ki67 levels. These findings suggest that Ficin-digested gelatin hydrolysate could be a beneficial candidate for novel breast cancer therapies.

MRI assessment of the bone adjacent to giant cell tumours and its association with local recurrence after intralesional curettage.

AIM: To assess the tumour border surrounding giant cell tumour of the bone (GCTB) using magnetic resonance imaging (MRI) and investigate its association with local recurrence. MATERIALS AND METHODS: Sixty-nine GCTBs in proximal tibiae and distal femurs were studied. The pathological basis of the paintbrush border sign was explored. Expression of Ki-67, matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), receptor activator of nuclear factor-κ B (RANK), and RANK ligand (RANKL) in GCTBs were investigated using immunohistochemistry. Patients treated with intralesional curettage were analysed retrospectively to investigate the prognostic role of the paintbrush border sign. The differences between rates were tested using the chi-square test or Fisher's exact test, as appropriate. RESULTS: The paintbrush border sign correlated well with infiltrative margins. The expression of MMP-9 was associated with the paintbrush border sign, and positively correlated with RANKL and VEGF expression. GCTBs with the paintbrush border sign had a higher rate of local recurrence (76.19 versus 20.59%, p<0.05). The paintbrush border sign was more common in proximal tibiae, and positively correlated with cystic change. The paintbrush border signs were detected at T1-weighted imaging, but the sign was only evident in four cases on T2-weighted imaging. CONCLUSION: Pathologically, the paintbrush border sign correlates well with invasion of the bone around GCTB. MMP-9 might play a key role in the formation of penetrating irregular margins. The paintbrush border sign is revealed as a risk factor for local recurrence of GCTB. Sagittal T1-weighted imaging is crucial to diagnose the paintbrush border sign.

Assessment of MMP-2/-9 expression by fluorescence endoscopy for evaluation of anastomotic healing in a murine model of anastomotic leakage.

BACKGROUND: Disturbance of intestinal wound closure leads to insufficient anastomotic healing and is associated with considerable morbidity following colorectal resections. Matrix metalloproteinases (MMPs) play a crucial role in regulation of wound closure. Here fluorescence endoscopy was evaluated for assessment of MMP-2/-9 expression during failed intestinal anastomotic healing. METHODS: Distal colonic anastomoses were performed as a model for disturbed healing in 36 Balb/c mice. Healing was evaluated endoscopically, macroscopically, and histologically after 1, 3 and 5 days. For detection of MMP-2/-9 expression fluorescence endoscopy (FE) was used following i.v.-administration of a Cy5.5-labeled MMP-2/-9 specific tracer. FE was complemented by quantification of the fluorescence signal using the MS-FX PRO Optical Imaging System. An overall leakage score was calculated and correlated with the results of FE. RESULTS: With increasing incidence of anastomotic leakage from POD1 (17%) to POD5 (83%) the uptake of the MMP tracer gradually increased (signal-to-noise ratio (SNR), POD1: 17.91 ± 1.251 vs. POD3: 30.56 ± 3.03 vs. POD5: 44.8 ± 4.473, P<0.0001). Mice with defective anastomotic healing showed significantly higher uptake compared to non-defective (SNR: 37.37± 3.63 vs. 26.16± 3.635, P = 0.0369). White light endoscopy and FE allowed evaluation of anastomotic healing and visualization of mucosal MMPs in vivo. Using FE based detection of MMPs in the anastomosis, an overall positive predictive value of 71.4% and negative predictive value of 66.6% was calculated for detection of anastomotic leakage. CONCLUSION: During disturbed anastomotic healing increased expression of MMP-2/-9 was observed in the anastomotic tissue. Fluorescence endoscopy for detection of MMP-2/-9 during the healing process might be a promising tool for early identification of anastomotic leakage.

Assessment of matrix metalloproteinase-8 and -9 levels in the peri-implant sulcular fluid among waterpipe (narghile) smokers and never-smokers with peri-implantitis.

OBJECTIVES: It is hypothesized that levels of matrix metalloproteinase (MMP)-8 and MMP-9 are significantly higher in the peri-implant sulcular fluid (PISF) of waterpipe-smokers (WS) compared with never-smokers with peri-implantitis. The aim of the present convenience sample case-control study was to compare the levels of MMP-8 and MMP-9 in the PISF of WS and never-smokers with peri-implantitis. MATERIAL AND METHODS: Individuals smoking waterpipe (Group 1) and never-smokers (Group 2) were included. Demographic data was collected using a questionnaire. Peri-implant probing depth (PPD) was measured and crestal bone loss (CBL) was measured on digital bitewing radiographs. PISF samples were collected using paper strips and the collected PISF volume was determined. Levels of MMP-8 and MMP-9 were measured using enzyme-linked immunosorbent assay. Study sample-size was estimated and statistical analysis was performed. p values < .05 were considered statistically significant. RESULTS: Sixty-six individuals (33 individuals in Group 1 and 33 in Group 2) were included. In Groups 1 and 2, 41 and 44 implants, respectively were placed. The mean total PPD (p < .001) and peri-implant CBL (p < .001) was statistically significantly higher around implants affected by peri-implantitis in Group 1 compared with Group 2. The PISF volume (p < .05) collected and levels of MMP-8 (p < .01) and MMP-9 (p < .01) were statistically significantly higher among individuals in Group 1 compared with Group 2. CONCLUSION: PISF levels of MMP-8 and MMP-9 are significantly higher among WS compared with never-smokers with peri-implantitis.

Quantitative Assessment of Invasion of High-Grade Gliomas Using Diffusion Tensor Magnetic Resonance Imaging.

OBJECTIVE: To determine heterogeneity of high-grade glioma (HGG) and its surrounding area and explore quantitative analysis of invasion of HGG using diffusion tensor imaging. METHODS: This study included 14 patients with HGG and preoperative magnetic resonance imaging and diffusion tensor imaging examinations. Three regions of interest were placed. Apparent diffusion coefficient (ADC) and fractional anisotropy (FA) values of these regions of interest were measured, and specimens from the 3 regions of interest were obtained under navigation guidance. Postoperative examinations of specimens were carried out. Correlations between ADC and FA values and tumor cell density were evaluated. RESULTS: Median survival was 36.7 months. As distance from the tumor increased, the number of tumor cells significantly decreased. Regarding levels of matrix metalloproteinase-9 and Ki-67, only the differences between tumor and distances of 1 cm and 2 cm away from the tumor were statistically significant. For analysis of the relationship between tumor cell density and ADC and FA values, the discriminant formulas were as follows: G1 = -13.678 + 14984.791 (X) + 14443.847 (Y) (tumor cell density ≥10%); G2 = -11.649 + 14443.847 (X) + 33.285 (Y) (tumor cell density <10%). CONCLUSIONS: We verified the heterogeneity of HGG and its surrounding area and found that patients with extensive resection may have longer survival. We also found a few formulas using FA and ADC values to predict tumor cell density.

Crosstalk Between Enzyme Matrix Metalloproteinases 2 and 9 and Regulatory T Cell Immunity in the Global Burden of Atherosclerosis.

Changes in immune and inflammatory responses may play a crucial role in the development and progression of atherosclerosis, as an autoimmune, chronic and progressive inflammatory disease. Immunological activity and vascular inflammation during atherosclerosis can be modulated by autoimmune responses against self-antigens, according to changeable risk factors (cholesterol, oxidized low-density lipoprotein (ox-LDL) in the vascular wall, fatty acids, etc.), and accompanied by accumulation of leucocytes and proinflammatory cytokines, which stimulate the transcription of matrix metalloproteinases (MMPs), whose concentration are increased in foam cell-rich regions. Regulatory T cells (Tregs) represent a unique subpopulation of T cells specialized in the regulation of immune response and in the suppression of proatherogenic T cells. The aim of our study was to examine the interactions between the concentration of enzyme matrix metalloproteinases 2 and 9 (MMP-2 and 9) in urine and the percentage of Tregs in peripheral blood of two groups of patients: with carotid artery stenosis (CAS), undergoing surgery and with mild atherosclerosis (A) from general practice. The method of enzyme immunoassay (ELISA) was used to determine enzyme MMP expression, and Tregs was examined by flow cytometric analysis. Our data have showed a large increase in the enzyme MMP-2 and 9 in the urine of CAS and A patients in comparison with healthy controls and indicated this method as an easy marker for the monitoring of the development of atherosclerosis. Simultaneously, the diminished number of Tregs in the same patients pointed the importance of these regulatory mechanisms in the etiopathogenesis of atherosclerosis and possible Tregs-mediated therapy.

Contractile efficiency of dystrophic mdx mouse muscle: in vivo and ex vivo assessment of adaptation to exercise of functional end points.

Progressive weakness is a typical feature of Duchenne muscular dystrophy (DMD) patients and is exacerbated in the benign mdx mouse model by in vivo treadmill exercise. We hypothesized a different threshold for functional adaptation of mdx muscles in response to the duration of the exercise protocol. In vivo weakness was confirmed by grip strength after 4, 8, and 12 wk of exercise in mdx mice. Torque measurements revealed that exercise-related weakness in mdx mice correlated with the duration of the protocol, while wild-type (WT) mice were stronger. Twitch and tetanic forces of isolated diaphragm and extensor digitorum longus (EDL) muscles were lower in mdx compared with WT mice. In mdx, both muscle types exhibited greater weakness after a single exercise bout, but only in EDL after a long exercise protocol. As opposite to WT muscles, mdx EDL ones did not show any exercise-induced adaptations against eccentric contraction force drop. qRT-PCR analysis confirmed the maladaptation of genes involved in metabolic and structural remodeling, while damage-related genes remained significantly upregulated and angiogenesis impaired. Phosphorylated AMP kinase level increased only in exercised WT muscle. The severe histopathology and the high levels of muscular TGF-ß1 and of plasma matrix metalloproteinase-9 confirmed the persistence of muscle damage in mdx mice. Therefore, dystrophic muscles showed a partial degree of functional adaptation to chronic exercise, although not sufficient to overcome weakness nor signs of damage. The improved understanding of the complex mechanisms underlying maladaptation of dystrophic muscle paves the way to a better managment of DMD patients.NEW & NOTEWORTHY We focused on the adaptation/maladaptation of dystrophic mdx mouse muscles to a standard protocol of exercise to provide guidance in the development of more effective drug and physical therapies in Duchenne muscular dystrophy. The mdx muscles showed a modest functional adaptation to chronic exercise, but it was not sufficient to overcome the progressive in vivo weakness, nor to counter signs of muscle damage. Therefore, a complex involvement of multiple systems underlies the maladaptive response of dystrophic muscle.

The assessment of matrix metalloproteinase-9 expression and angiogenesis in colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent types of cancer in the world. Between tumor cells and the stroma mutual interconnections are established that favors the tumor development and metastasis. In this respect, the extracellular matrix is remodeled so that it may become totally different from a morphologic perspective than the stroma of the organ in which the tumor develops. Matrix metalloproteinases (MMPs) have an essential role in the remodeling of the tumor stroma. We assessed the expression of MMP-9 on a number of 31 stage III colorectal adenocarcinomas. Generally, MMP-9 had a high but inconstant expression in tumor cells. The highest expression was found in poorly and moderately differentiated carcinomas, with a lower expression in well-differentiated colorectal cancers. Occasionally, MMP-9 expression was identified also in peritumoral macrophages and in stromal cells. Metastasis-free lymph nodes had an intense positive reaction in both macrophages and lymphocytes. The intensely positive reaction was observed for the macrophages and lymphocytes in the tumor necrosis regions. The process of angiogenesis was generally correlated with the intensity of MMP-9 reaction.

Klotho Reduction in Alveolar Macrophages Contributes to Cigarette Smoke Extract-induced Inflammation in Chronic Obstructive Pulmonary Disease.

Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Klotho, an anti-aging protein, has an anti-inflammatory function. However, the role of Klotho has never been investigated in COPD. The aim of this study is to investigate the possible role of Klotho by alveolar macrophages in airway inflammation in COPD. Klotho levels were assessed in the lung samples and peripheral blood mononuclear cells of non-smokers, smokers, and patients with COPD. The regulation of Klotho expression by cigarette smoke extract (CSE) was studied in vitro, and small interfering RNA (siRNA) and recombinant Klotho were employed to investigate the role of Klotho on CSE-induced inflammation. Klotho expression was reduced in alveolar macrophages in the lungs and peripheral blood mononuclear cells of COPD patients. CSE decreased Klotho expression and release from MH-S cells. Knockdown of endogenous Klotho augmented the expression of the inflammatory mediators, such as MMP-9, IL-6, and TNF-α, by MH-S cells. Exogenous Klotho inhibited the expression of CSE-induced inflammatory mediators. Furthermore, we showed that Klotho interacts with IκBα of the NF-κB pathway. Dexamethasone treatment increased the expression and release level of Klotho in MH-S cells. Our findings suggest that Klotho plays a role in sustained inflammation of the lungs, which in turn may have therapeutic implications in COPD.

The role of MMP-2, MMP-9, and TIMP-1 in the pathogenesis of nasal polyps: Immunohistochemical assessment at eight different levels in the epithelial, subepithelial, and deep layers of the mucosa.

We conducted a prospective study to investigate the role of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the pathogenesis of nasal polyps. Our study group consisted of 24 patients-21 men and 3 women, aged 23 to 70 years (mean: 45.97 ± 11.60)-with nasal polyposis who underwent functional endoscopic sinus surgery. For comparison purposes, we assembled a control group of 11 patients-6 men and 5 women, aged 18 to 56 years (mean: 29.90 ± 14.22)-without nasal polyps who underwent septoplasty and/or rhinoplasty. We analyzed 36 polyp specimens obtained from the study group (10 from the nasal cavity, 10 from the maxillary sinus, and 16 from the ethmoid sinus) and 11 tissue specimens from the control group (each control provided 1 specimen from the inferior turbinate). We then calculated the mean number of these cells in the epithelium, subepithelial layer of the lamina propria, and the deep paraglandular layer of the mucosa. In general, we found that MMP-2, MMP-9, and TIMP-1 values were higher in the nasal polyp group. These differences became less so as patients' ages and the duration of polyps increased. We conclude that the most important role that MMP-2 plays in polyp growth may be in terms of perivascular localization and an increase in vascular permeability, which causes inflammatory cell migration and edema in the extracellular matrix. An increase in MMP-2 in glandular tissue may lead to hydrolysis of tissue matrix components. The degraded extracellular matrix may result in fibrosis of the polyps. An increase of MMP-9 in the apical part of the epithelium in the polypoid tissue of the nasal cavity, maxillary sinus, and ethmoid sinus may facilitate the epithelial and endothelial cell migration that is observed during polyp development and growth.

New tools for the quantitative assessment of prodrug delivery and neurotoxicity.

Systemic off-target toxicities, including neurotoxicity, are prevalent side effects in cancer patients treated with a number of otherwise highly efficacious anticancer drugs. In the current study, we have: (1) developed a new analytical metric for the in vivo preclinical assessment of systemic toxicities/neurotoxicity of new drugs and delivery systems; and (2) evaluated, in mice, the in vivo efficacy and toxicity of a versatile and modular NanoDendron (ND) drug delivery and imaging platform that we recently developed. Our paclitaxel-carrying ND prodrug, ND(PXL), is activated following proteolytic cleavage by MMP9, resulting in localized cytotoxic chemotherapy. Using click chemistry, we combined ND(PXL) with a traceable beacon, ND(PB), yielding ND(PXL)-ND(PB) that functions as a theranostic compound. In vivo fluorescence FRET imaging of this theranostic platform was used to confirm localized delivery to tumors and to assess the efficiency of drug delivery to tumors, achieving 25-30% activation in the tumors of an immunocompetent mouse model of breast cancer. In this model, ND-drug exhibited anti-tumor efficacy comparable to nab-paclitaxel, a clinical formulation. In addition, we combined neurobehavioral metrics of nociception and sensorimotor performance of individual mice to develop a novel composite toxicity score that reveals and quantifies peripheral neurotoxicity, a debilitating long-term systemic toxicity of paclitaxel therapy. Importantly, mice treated with nab-paclitaxel developed changes in behavioral metrics with significantly higher toxicity scores indicative of peripheral neuropathy, while mice treated with ND(PXL) showed no significant changes in behavioral responses or toxicity score. Our ND formulation was designed to be readily adaptable to incorporate different drugs, imaging modalities and/or targeting motifs. This formulation has significant potential for preclinical and clinical tools across multiple disease states. The studies presented here report a novel toxicity score for assessing peripheral neuropathy and demonstrate that our targeted, theranostic NDs are safe and effective, providing localized tumor delivery of a chemotherapeutic and with reduced common neurotoxic side-effects.

Assessment of Nogo-66 receptor 1 function in vivo after spinal cord injury.

BACKGROUND: Neuronal Nogo-66 receptor 1 (NgR1) has attracted attention as a converging point for mediating the effects of myelin-associate inhibitory ligands in the central nervous system, establishing the growth-restrictive environment, and limiting axon regeneration after traumatic injury. OBJECTIVE: To investigate the factors that may be contributing to the discrepancy in the importance of NgR1, which has been undermined by several studies that have shown the lack of substantial axon regeneration after spinal cord injury (SCI) in NgR1-knockout or -knockdown animal models. METHODS: We used mice carrying either a homozygous or heterozygous null mutation in the NgR1 gene and subjected them to either a moderate or severe SCI. RESULTS: Locomotor function assessments revealed that the level of functional recovery is affected by the degree of injury suffered. NgR1 ablation enhanced local collateral sprouting in the mutant mice. Reactive astrocytes and chondroitin sulfate proteoglycans (CSPGs) are upregulated surrounding the injury site. Matrix metalloproteinase-9, which has been shown to degrade CSPGs, was significantly upregulated in the homozygous mutant mice compared with the heterozygous or wild-type mice. However, CSPG levels remained higher in the homozygous compared with the heterozygous mice, suggesting that CSPG-degrading activity of matrix metalloproteinase-9 may require the presence of NgR1. CONCLUSION: Genetic ablation of NgR1 may lead to significant recovery in locomotor function after SCI. The difference in locomotor recovery we observed between the groups that suffered various degrees of injury suggests that injury severity may be a confounding factor in functional recovery after SCI.

The assessment of the relationship between amniotic fluid matrix metalloproteinase-9 and zinc levels with adverse obstetric outcomes.

OBJECTIVE: To examine the relationship between the amniotic fluid MMP-9 and zinc levels during 16-19th gestational weeks and perinatal outcomes. METHOD: One hundred and seventeen singleton pregnancies that underwent genetic amniocentesis from January 2005 through November 2009 were evaluated. Subjects were divided into two main groups: a control group (group 1) (n: 74), and an adverse obstetric outcomes group (group 2) (n: 43). Group 2 consisted of the following: preterm birth group, gestational hypertension and preeclampsia group, gestational diabetes group, fetal growth restriction group, macrosomia group, and pregnancy loss group. MMP-9 and zinc (Zn) values in the amniocentesis materials sampled between the 16th and 19th gestational weeks were analyzed retrospectively in terms of perinatal outcomes. Any significant difference among the groups was assessed by unpaired samples t test and the Mann-Whitney U test. Statistical significance was defined as p < 0.05. RESULTS: A comparison among groups showed no significant difference in terms of Zn results between the group 1 and 2 (p = 0.879). MMP-9 levels were significantly lower in both the preterm birth group (p = 0.043) and group 1 (p = 0.015). CONCLUSION: We found that the amniotic fluid MMP-9 levels of patients who delivered preterm were significantly lower between the 16th and 19th gestational weeks.