[Clinical analysis of three-dimensional surgical planning system for guiding robot-assisted selective artery clamping partial nephrectomy in completely endophytic renal tumor].

Objective: To examine the safety and feasibility of three-dimensional (3D) surgical planning system for guiding robot-assisted selective artery clamping partial nephrectomy (RASPN) in completely endophytic renal tumor. Methods: Clinical data of 32 patients who suffered from completely endophytic renal tumor and underwent RASPN associated with 3D surgical planning system in Department of Urology, Sun Yat-Sen University Cancer Center from November 2018 to August 2021 were analyzed retrospectively. There were 21 males and 11 females, with the age (M (IQR)] of 45.0 (17.5) years (range: 30 to 68 years). Fifteen tumors were located on the left and 17 on the right. Maximum tumor diameter, R.E.N.A.L. Score and preoperative estimated glomerular filtration rate (eGFR) were 27.5 (13.0) mm (range: 14 to 50 mm), 10.0 (1.8) (range: 7 to 11), and 105.5 (15.7) ml·min-1·(1.73 m2)-1 (range: 71.1 to 124.8 ml·min-1·(1.73 m2)-1), respectively. The 3D reconstruction before RASPN was performed in all patients to formulate surgical planning, mainly including stereo localization of renal mass, confirmation of tumor feeding artery, and injury prediction of collecting system or vessel via "2 mm distance method" defined as probable damage of renal pelvis/calyx and artery/vein when these tissues were less than 2 mm away from tumor. Results: Totally 32 patients successfully underwent RASPN guided by 3D surgical planning system, without conversion to open operation or radical nephrectomy. Rapid location of tumor and selective clamping of artery were achieved in all cases and no one encountered global ischemia, with branch occlusion time of 24.5 (15.4) min (range: 12 to 60 min) and coincidence rate of 95.0% (57/60) between planned and actual clamping vessels. The sensitivity and specificity of 2 mm distance method for predicting the injury of collecting system were 13/15 and 17/17, respectively. The operating time of 185 (48) minuetes (range: 76 to 295 minutes) and estimated blood loss of 200 (350) ml (range: 20 to 800 ml) were observed, without intraoperative transfusion case. There was one patient performed with renal vein repair. Clavien-Dindo postoperative grade Ⅱ and Ⅲa bleeding complications occurred in 2 cases, and no postoperative urinary fistula was found. The length of hospitalization was 3 (0) days (range: 2 to 10 days). The pathological diagnosis demonstrated 4 chromophobe cell carcinomas and 2 angiomyolipomas, besides 26 clear cell carcinomas including one positive surgical margin. The postoperative latest eGFR was 103.9(18.5) ml·min-1·(1.73 m2)-1 (range: 75.8 to 122.3 ml·min-1·(1.73 m2)-1) and no tumor recurrence or metastasis was detected during the follow-up time of 15.4 (13.9) months (range: 3 to 35 months). Conclusion: For RASPN in completely endophytic renal tumor, 3D surgical planning system is contributed to determining mass position, defining tumor feeding artery, and predicting collecting system/vessel injury, which benefited precise tumor resection, postoperative renal function preservation, and perioperative urinary fistula and bleeding complication decrease.

Bedaquiline for multidrug-resistant TB in paediatric patients.

BACKGROUND: TMC207-C211 (NCT02354014) is a Phase 2, open-label, multicentre, single-arm study to evaluate pharmacokinetics, safety/tolerability, antimycobacterial activity and dose selection of bedaquiline (BDQ) in children (birth to <18 years) with multidrug-resistant-TB (MDR-TB).METHODS: Patients received 24 weeks' BDQ with an anti-MDR-TB background regimen (BR), followed by 96 weeks of safety follow-up. Results of the primary analysis are presented based on data up to 24 weeks for Cohort 1 (≥12-<18 years; approved adult tablet at the adult dosage) and Cohort 2 (≥5-<12 years; age-appropriate 20 mg tablet at half the adult dosage).RESULTS: Both cohorts had 15 patients, of whom respectively 53% and 40% of Cohort 1 and Cohort 2 children had confirmed/probable pulmonary MDR-TB. Most patients completed 24 weeks´ BDQ/BR treatment (Cohort 1: 93%; Cohort 2: 67%). Geometric mean BDQ area under the curve 168h values of 119,000 ng.h/mL (Cohort 1) and 118,000 ng.h/mL (Cohort 2) at Week 12 were within 60-140% (86,200-201,000 ng.h/mL) of adult target values. Few adverse event (AE) related discontinuations or serious AEs, and no QTcF >460 ms during BDQ/BR treatment or deaths occurred. Of MGIT-evaluable patients, 6/8 (75%) Cohort 1 and 3/3 (100%) Cohort 2 culture converted.CONCLUSION: In children and adolescents aged ≥5-<18 years with MDR-TB, including pre-extensively drug-resistant-TB (pre-XDR-TB) or XDR-TB, 24 weeks of BDQ provided a comparable pharmacokinetic and safety profile to adults.

[Changes of immunohistochemical biomarkers before and after neoadjuvant chemotherapy in breast cancer and their prognosis].

Objective: To investigate changes in the expression of immunohistochemical (IHC) markers and factors associated with the effect of chemotherapy before and after neoadjuvant chemotherapy (NAC). Methods: A retrospective study included 200 breast cancer patients treated with NAC between January 2016 and December 2018. We analyzed the changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67 in pre- and post-treated samples and the predictive factors of NAC. Results: Among the 200 cases, 16 cases were luminal A, 108 cases were luminal B, 36 cases were HER2+subtype, and 40 cases were basal-like. Twenty-five patients (12.50%) achieved pathological complete remission (PCR).There were significant differences in PR and Ki-67 before and after NAC but there were no differences in ER and HER2.In univariate analysis, factors associated with PCR were tumor less than 5 cm(P=0.009), non-luminal breast cancer (P=0.001), ER negative(P=0.001), PR negative (P=0.029) and HER2 positive(P=0.001). Tumor less than 5 cm [P=0.020, OR=2.581, 95%CI (1.207, 5.753)], ER negative [P=0.011, OR=2.264, 95%CI (1.207, 4.248)] and HER2 positive[P=0.007, OR=2.412, 95%CI (1.275, 4.561)] remained predictive variables in multivariate analysis after correction for the other variables. Conclusions: The expression of Ki-67 decreases after NAC. Negative PR and ER and positive HER2 status are related to the efficacy of pCR for breast cancer, and have guiding significance for the prognosis evaluation of NAC.

Formation and Development of Environmental Forensics System in China.

ABSTRACT: With the growing attention on ecological environment problems and gradual realization of ecological environment value, environmental damage has jumped from administrative penalty to a new stage, judicial penalty, and environmental damage appraisal has provided a legal weapon to safeguard ecological security. As a new forensic category of China with high comprehensiveness and technical difficulty, environmental damage appraisal involves diversified and complex subjects, fields and appraisal objects, and is still in an early stage in terms of theory and practice. This study aims to provide an important reference for the improvement of the Chinese environmental damage appraisal system of environmental damage by summarizing advanced international experience in areas such as laws and regulations, working mechanism and technical system, and putting forward targeted countermeasures and suggestions based on the problems existing in the development and practice of environmental damage appraisal in China.

Evaluation of a second-generation monoenergetic reconstruction algorithm for lesion contrast and venous invasion in pancreatic ductal adenocarcinomas.

OBJECTIVE: At lower energy levels, virtual monochromatic imaging by dual-energy computed tomography improves lesion attenuation but produces greater image noise with the conventional monoenergetic reconstruction algorithm (Mono). Recently, a second-generation algorithm (Mono+) was introduced to overcome this limitation. We compared the quality of images obtained with these algorithms and investigated the optimal energy selection for pancreatic ductal adenocarcinomas (PDACs). PATIENTS AND METHODS: Image data from 54 PDAC cases were generated at 40, 50, 60, 70, and 80 keV using Mono and Mono+. Image quality was objectively assessed by comparing the signal-to-noise ratios (SNRs), noise, and the contrast-to-noise ratios (CNRs) at different keV levels and between these algorithms at the same keV level. Lesion conspicuity and venous invasion were subjectively assessed. RESULTS: For Mono, the mean pancreas and tumour SNRs peaked at 70 keV (p<0.001). The noise increased as the energy level decreased (p<0.001). CNRtumour remained unchanged. For Mono+, the mean pancreas SNR peaked at 40 keV (p<0.001). The mean tumour SNR and noise remained unchanged. The tumour CNRs were highest at 40 keV (4.9 times the CNR of Mono 40 keV, p<0.001). Subjectively, lesion conspicuity was best at Mono+ 40 keV (p<0.001) and it showed higher diagnostic performance levels on venous invasion assessment against Mono. CONCLUSIONS: Mono+ produced better image quality, and 40 keV is recommended for the diagnosis of PDAC.

Application of Nonlinear Optical Microscopic Imaging Technology for Quality Assessment of Donor Kidneys.

BACKGROUND: Nonlinear optical microscopic (NLOM) imaging technique shows its high resolution imaging features in histocytology. The purpose of this study was to investigate NLOM imaging technique as a useful tool for a donor kidney quality assessment. MATERIALS AND METHODS: Eighty-three pretransplant kidney biopsies from adult donors were analyzed retrospectively. Each specimen was paraffin-embedded and sectioned into 2 consecutive 5-µm thick sections. One section was stained with Masson trichrome, and the other was left unstained for NLOM imaging using second harmonic generation combined with two-photon excited fluorescence (SHG/TPEF). The pretransplant kidney quality was assessed by an experienced pathologist using the Remuzzi scoring system, which characterizes renal tissue morphology into 4 aspects: tubular atrophy, interstitial fibrosis, glomerulosclerosis, and vascular injury. The K coefficient was used to measure the consistency of the Remuzzi scores between conventional Masson trichrome stained images and SHG/TPEF images. RESULTS: NLOM imaging technology can capture high-resolution tissue images from unstained renal tissue, is easy to operate, and shortens time-consuming histological processing procedures. No significant differences (P > .05) were found between the Remuzzi scores of the SHG/TPEF images and the Masson trichrome stained images. The high κ coefficients (0.804-0.895) showed a good consistency between these 2 techniques. CONCLUSION: The NLOM technique is suitable for renal tissue imaging and could potentially be used for routine pretransplant kidney evaluation in clinical settings.

Long-term quality of life measured by the University of Washington QoL questionnaire (version 4) in patients with oral cancer treated with or without reconstruction with a microvascular free flap.

We used the University of Washington Quality of Life (UW-QoL) questionnaire (version 4) to assess the long-term quality of life (QoL) of patients with oral squamous cell carcinoma treated with or without reconstruction with a microvascular free flap, and all patients with T2-T4 oral squamous cell carcinoma (SCC) treated in this way were eligible for the study. A total of 139 patients' personal details, medical history, and QoL scores were collected and analysed. The mean (SD) overall QoL score was 73.09 (14.8) for patients with T2-T4 oral SCC. The mean (SD) global QoL scores of patients who had had reconstructions was 75.68 (13.85) and of patients who had not 71.00 (15.34) (t=1.864, df=137, p=0.064). Univariate and multivariate analyses indicated that site of tumour, T stage, and the need for postoperative radiotherapy had significant effects on the global QoL scores. Among patients with T2 oral SCC there was no significant difference between patients who did and did not have reconstructions. However, there were significant differences among patients with T3/T4 SCC in scores for appearance, recreation, mood, anxiety, chewing, swallowing, and speech depending on whether they did or did not have reconstructions. There were significant differences in the domains recreation, chewing, speech, and taste, depending on the primary site of the tumour. We conclude that reconstruction with a microvascular free flap had a beneficial effect on the treatment of SCC and improved the QoL of patients with oral SCC.

Second-harmonic imaging microscopy for identifying colorectal intraepithelial neoplasia.

In this study, second-harmonic imaging microscopy was used to monitor precancerous colorectal lesions at different stages. It was found that the morphology of glands and lamina propria in mucosa changes with the progression of colorectal diseases from normal to low-grade intraepithelial neoplasia to high-grade intraepithelial neoplasia and this microscopy has the ability of direct visualization of these warning symptoms. Furthermore, two morphologic variables were quantified to determine the changes of glands and collagen in lamina propria during the development of colorectal intraepithelial neoplasia. These results suggest that second-harmonic imaging microscopy has the potential in label-freely and effectively distinguishing between normal and precancerous colorectal tissues, and will be helpful for early diagnosis and treatment of colorectal diseases.

Household air pollution and personal exposure to nitrated and oxygenated polycyclic aromatics (PAHs) in rural households: Influence of household cooking energies.

Residential solid fuels are widely consumed in rural China, contributing to severe household air pollution for many products of incomplete combustion, such as polycyclic aromatic hydrocarbons (PAHs) and their polar derivatives. In this study, concentrations of nitrated and oxygenated PAH derivatives (nPAHs and oPAHs) for household and personal air were measured and analyzed for influencing factors like smoking and cooking energy type. Concentrations of nPAHs and oPAHs in kitchens were higher than those in living rooms and in outdoor air. Exposure levels measured by personal samplers were lower than levels in indoor air, but higher than outdoor air levels. With increasing molecular weight, individual compounds tended to be more commonly partitioned to particulate matter (PM); moreover, higher molecular weight nPAHs and oPAHs were preferentially found in finer particles, suggesting a potential for increased health risks. Smoking behavior raised the concentrations of nPAHs and oPAHs in personal air significantly. People who cooked food also had higher personal exposures. Cooking and smoking have a significant interaction effect on personal exposure. Concentrations in kitchens and personal exposure to nPAHs and oPAHs for households using wood and peat were significantly higher than for those using electricity and liquid petroleum gas (LPG).

Label-free detection of fibrillar collagen deposition associated with vascular elements in glioblastoma multiforme by using multiphoton microscopy.

Glioblastoma multiforme (GBM-WHO grade IV) is the most common and the most aggressive form of brain tumors in adults with the median survival of 10-12 months. The diagnostic detection of extracellular matrix (ECM) component in the tumour microenvironment is of prognostic value. In this paper, the fibrillar collagen deposition associated with vascular elements in GBM were investigated in the fresh specimens and unstained histological slices by using multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Our study revealed the existence of fibrillar collagen deposition in the adventitia of remodelled large blood vessels and in glomeruloid vascular structures in GBM. The degree of fibrillar collagen deposition can be quantitatively evaluated by measuring the adventitial thickness of blood vessels or calculating the ratio of SHG pixel to the whole pixel of glomeruloid vascular structure in MPM images. These results indicated that MPM can not only be employed to perform a retrospective study in unstained histological slices but also has the potential to apply for in vivo brain imaging to understand correlations between malignancy of gliomas and fibrillar collagen deposition.

Differentiating the two main histologic categories of fibroadenoma tissue from normal breast tissue by using multiphoton microscopy.

Multiphoton microscopy has become a novel biological imaging technique that allows cellular and subcellular microstructure imaging based on two-photon excited fluorescence and second harmonic generation. In this work, we used multiphoton microscopy to obtain the high-contrast images of human normal breast tissue and two main histologic types of fibroadenoma (intracanalicular, pericanalicular). Moreover, quantitative image analysis was performed to characterize the changes of collagen morphology (collagen content, collagen orientation). The results show that multiphoton microscopy combined with quantitative method has the ability to identify the characteristics of fibroadenoma including changes of the duct architecture and collagen morphology in stroma. With the advancement of multiphoton microscopy, we believe that the technique has great potential to be a real-time histopathological diagnostic tool for intraoperative detection of fibroadenoma in the future.

The risk of bacteria in foam sclerotherapy: does the condition of the air in outpatient vs. operating rooms make a difference?

BACKGROUND: Foam sclerotherapy has been one of the most frequently applied therapeutic methods for the treatment of varicose veins and venous malformation. Few studies have investigated the sterile condition of the air used for foam creation. OBJECTIVES: To evaluate the bacterial conditions in foam created from the air in outpatient and operating rooms used for sclerotherapy treatment, in order to provide the basis for clinical applications in foam sclerotherapy. MATERIALS AND METHODS: In this study, we measured the amount of airborne microorganisms in the air in the operating room, and in the outpatient room, the polidocanol (POL) foam created using the air in the respective rooms, and the microorganisms present in the liquid POL. We used the settle plate method for air sampling. We used the Tessari method to generate foam. The experiment was repeated three times. RESULTS: Typical bacterial colonies were formed on nutrient agar plates in both the outpatient room and operating room air groups. No colonies were cultivated in the liquid POL. Although the use of sterile or nonsterile air in sclerotherapy produces significantly different results in the counts of colony-forming units, there was no difference in the foam created using sterile or nonsterile air (i.e. no colonies were cultivated on either foam). CONCLUSIONS: There is no increased risk of infection when using foam sclerotherapy in outpatient rooms compared with using foam created under sterile operating-room conditions.

Monitoring wound healing of elastic cartilage using multiphoton microscopy.

OBJECTIVE: To demonstrate the ability of multiphoton microscopy (MPM) for monitoring wound healing of elastic cartilage. METHOD: In a rabbit ear model, four cartilage specimen groups at 1-day, 1-, 4-, 20-week healing time points as well as a normal elastic cartilage were examined with MPM without using labeling agents. MPM images at wound margins were obtained from specimens at different healing stages, compared with the Hematoxylin and Eosin (H&E) stained images. Image analysis was performed to characterize the collagen morphology for quantifying the wound healing progression of elastic cartilage. RESULTS: MPM provided high-resolution images of elastic cartilage at varying depths. Comparisons of the images of specimens at different healing stages show obvious cell growth and matrix deposition. The results are consistent with the histological results. Moreover, quantitative analysis results show significant alteration in the collagen cavity size or collagen orientation index during wound healing of elastic cartilage, indicating the possibility to act as indicators for monitoring wound healing. CONCLUSION: Our results suggested that MPM has the ability to monitor the wound healing progression of elastic cartilage, based on the visualization of cell growth and proliferation and quantitative characterization of collagen morphology during wound healing.

Nonlinear spectral imaging of human hypertrophic scar based on two-photon excited fluorescence and second-harmonic generation.

BACKGROUND: A noninvasive method using microscopy and spectroscopy for analysing the morphology of collagen and elastin and their biochemical variations in skin tissue will enable better understanding of the pathophysiology of hypertrophic scars and facilitate improved clinical management and treatment of this disease. OBJECTIVE: To obtain simultaneously microscopic images and spectra of collagen and elastin fibres in ex vivo skin tissues (normal skin and hypertrophic scar) using a nonlinear spectral imaging method, and to compare the morphological structure and spectral characteristics of collagen and elastin fibres in hypertrophic scar tissues with those of normal skin, to determine whether this approach has potential for in vivo assessment of the pathophysiology of human hypertrophic scars and for monitoring treatment responses as well as for tracking the process of development of hypertrophic scars in clinic. METHODS: Ex vivo human skin specimens obtained from six patients aged from 10 to 50 years old who were undergoing skin plastic surgery were examined. Five patients had hypertrophic scar lesions and one patient had no scar lesion before we obtained his skin specimen. A total of 30 tissue section samples of 30 mum thickness were analysed by the use of a nonlinear spectral imaging system consisting of a femtosecond excitation light source, a high-throughput scanning inverted microscope, and a spectral imaging detection system. The high-contrast and high-resolution second harmonic generation (SHG) images of collagen and two-photon excited fluorescence (TPEF) images of elastin fibres in hypertrophic scar tissues and normal skin were acquired using the extracting channel tool of the system. The emission spectra were analysed using the image-guided spectral analysis method. The depth-dependent decay constant of the SHG signal and the image texture characteristics of hypertrophic scar tissue and normal skin were used to quantitatively assess the amount, distribution and orientation of their collagen and elastin components. RESULTS: Our experiments and data analyses demonstrated apparent differences between hypertrophic scar tissue and normal skin in terms of their morphological structure and the spectral characteristics of collagen and elastin fibres. These differences can potentially be used to distinguish hypertrophic scar tissues from normal skin and to evaluate treatment responses. CONCLUSIONS: All the measurements were performed in backscattering geometry and demonstrated that nonlinear spectral imaging has the ability to differentiate hypertrophic scar tissue from normal skin based on noninvasive SHG imaging, and TPEF imaging revealed the microstructure and spectral features of collagen and elastin fibres. With the advances in spectral imaging apparatus miniaturization, we have good reason to believe that this approach can become a valuable tool for the in vivo pathophysiology study of human skin hypertrophic scars and for assessing the treatment responses of this disfiguring disease in clinic. It can also be used to track the development of hypertrophic scars and to study wound healing processes in a noninvasive fashion without biopsy, fixation, sectioning and the use of exogenous dyes or stains.

Modulation of human cytomegalovirus immediate-early gene enhancer by mitogen-activated protein kinase kinase kinase-1.

The immediate-early (IE) promoter of human cytomegalovirus (HCMV) constitutes a primary genetic switch, which determines the progression of viral infection. Earlier reports by others have shown mitogen-activated protein kinase kinase kinase-1 (MEKK1) to be able to up-regulate HCMV-IE promoter through downstream mitogen-activated protein kinase (MAPK) pathways. However, we noticed that the activation of the HCMV-IE promoter by constitutively active MEKK1 (MEKK1-TRU) might not be through the MAPK pathways. Using a HCMV-IE enhancer/promoter (- 522 to + 72) driving a luciferase reporter, we demonstrated that the downstream MAPK activation actually repressed the up-regulation of the promoter by MEKK1 in CHO-K1 and human 293 cells. We further found that the up-regulation of HCMV-IE promoter by MEKK1 could be in great extent suppressed by over-expression of IkappaBalpha. Deletion of the NFkappaB/rel sites in the HCMV-IE enhancer region by mutagenesis proportionally reduced the transcriptional activation by MEKK1-TRU, whereas deletion of the ATF/CREB binding sites or cyclic AMP response elements (CRE) had no effects. Furthermore, the NFkappaB/rel deletion mutant also showed repression on the basic transcription activity of the HCMV-IE promoter. Our results indicate that the NFkappaB/rel sites are not only responsible for the modulation of HCMV-IE enhancer activity by MEKK1 but also control the basic transcription activity of the HCMV-IE promoter. On the other hand, the four consensus CRE sites were found to have no function in the activation of the promoter by MEKK1.

Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells.

AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P<0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P < 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.

Nonproteolytic role for the urokinase receptor in cellular migration in vivo.

The urokinase receptor (uPAR) binds and localizes urokinase activity at cellular surfaces, facilitating fibrinolysis and cellular migration at sites of tissue injury. uPAR also participates in cellular signaling and regulates integrin-dependent adhesion and migration in vitro. We now report evidence that uPAR occupancy regulates cellular migration in vivo in the absence of functional urokinase. Recombinant murine KC (1.5 microg), a potent neutrophil chemoattractant, was delivered to the lungs of wild-type, urokinase-deficient or uPAR-deficient mice 18 h after intraperitoneal injection of 200 microg human immunoglobulin G (IgG) or a fusion protein composed of an amino-terminal receptor-binding fragment of urokinase and a human IgG Fc fragment (GFD-Fc). Whole lung lavage for recovery of leukocytes was performed 4 h later. KC treatment resulted in a 100-fold increase in lavage neutrophils. GFD-Fc injection resulted in >50% reduction in neutrophil influx in both wild-type and urokinase-deficient animals but had no effect on uPAR -/- mice. A concomitant reduction in alveolar protein leakage but no change in numbers of circulating neutrophils accompanied this attenuated inflammatory response. The reduction in neutrophil influx induced by GFD-Fc is thus related to uPAR occupancy and yet not due to disruption of uPAR-mediated proteolysis. These observations verify that protease-independent functions of uPAR operate in vivo and identify uPAR as a potential target for regulation of inflammatory processes characterized by neutrophil-mediated injury.

Urokinase receptor antagonists: discovery and application to in vivo models of tumor growth.

Urokinase receptor antagonists based on the growth factor domains of both human and murine urokinase which show sub-nanomolar affinities for their homologous receptors have been expressed as recombinant proteins. Further modification of these molecules by preparing fusions with the constant region of human IgG has led to molecules with high affinities and long in vivo half-lives. Smaller peptidic inhibitors have been obtained by a combination of bacteriophage display and peptide analog synthesis. All of these molecules inhibit the binding of the growth factor domain of uPA to the uPA receptor and enhance binding of the uPA receptor to vitronectin. Protein uPA receptor antagonists were tested in an in vivo tumor model using the human breast carcinoma MDAmb231 in immunodeficient mice. Both human and murine receptor antagonists showed significant inhibition of primary tumor growth, demonstrating that in vivo, both tumor and stromal cell uPA receptor dependent plasminogen activation can modulate tumor growth.