Development of a MET-targeted single-chain antibody fragment as an anti-oncogene targeted therapy for breast cancer.

The usage of monoclonal antibodies (mAbs) and antibody fragments, as a matter associated with the biopharmaceutical industry, is increasingly growing. Harmonious with this concept, we designed an exclusive modeled single-chain variable fragment (scFv) against mesenchymal-epithelial transition (MET) oncoprotein. This scFv was newly developed from Onartuzumab sequence by gene cloning, and expression using bacterial host. Herein, we examined its preclinical efficacy for the reduction of tumor growth, invasiveness and angiogenesis in vitro and in vivo. Expressed anti-MET scFv demonstrated high binding capacity (48.8%) toward MET-overexpressing cancer cells. The IC50 value of anti-MET scFv against MET-positive human breast cancer cell line (MDA-MB-435) was 8.4 µg/ml whereas this value was measured as 47.8 µg/ml in MET-negative cell line BT-483. Similar concentrations could also effectively induce apoptosis in MDA-MB-435 cancer cells. Moreover, this antibody fragment could reduce migration and invasion in MDA-MB-435 cells. Grafted breast tumors in Balb/c mice showed significant tumor growth suppression as well as reduction of blood-supply in response to recombinant anti-MET treatment. Histopathology and immunohistochemical assessments revealed higher rate of response to therapy. In our study, we designed and synthetized a novel anti-MET scFv which could effectively suppress MET-overexpressing breast cancer tumors.

Effects of exosomes derived from fibroblast cells on skin wound healing in Wistar rats.

BACKGROUND: The role of exosomes in areas, such as skin wound healing, have been of consideratble interest recently. However, the effects of exosomes derived mainly from fibroblast cells on wound healing have yet to be documented well. The study aimed to evaluate the effects of exosomes derived from fibroblast cells on wound healing in Wistar rats. METHODS: Human fetal skin was isolated afterward centrifuge, and trypsin 0.1% was added to the cells after removing DPBS from the Falcon tube, and the trypsin was removed. The cells were moved to culture flasks. Then, the secondary culture of Human Fetal Skin Fibroblast was done. The pellets containing exosomes were suspended in PBS, and to achieve purified exosomes, the suspended Exosome were passed through a 0.22 µm filter. The exosome solution was kept at - 20 ºC. In the in vivo phase, 48 male Wistar rats were divided into four groups. Group I, low-dose exosome (LDE) solution (150 µl/day), group II high-dose exosome (HDE) solution (300 µl/day), group III commercially available ointment (positive control (PC)) was topically applied on wounds and group VI without treatment (negative control (NC)). A skin biopsy was taken for histopathological analysis. Wound area, depth of ulcer, degree of granulation, and inflammation were assessed. For histopathological assessment, re-epithelialization, inflammatory cells, granulation tissue, crust formation, and collagen maturation (fibrosis) parameters were evaluated. RESULTS: Forty-eight male Wistar rats were included. The HDE group's showed accelerated healing compared to the NC and PC groups at 9 and 12 days. Inflammation and granulation were higher in the HDE, LDE, and PC groups than in the NC group (p < 0.05). The onset of re-epithelialization and collagen deposition was higher in the LDE, HDE, and PC groups, then on nine and 12-day, gradually maturing and extending through the ulcer (p < 0.05). On day 12, in almost all parameters, the LDE and HDE groups showed improved results compared to NC cases (p < 0.05). CONCLUSIONS: The results showed that the utilization of fibroblast-Exo significantly promoted cutaneous wound healing in a rat full-thickness skin ulcer model. This is a potential innovation for cell-free therapy from fibroblast-Exo as a closed structure similar to human cells.

Comparison of the effects of human fetal umbilical cord-derived hyaluronic acid and fibroblast-derived exosomes on wound healing in rats.

INTRODUCTION: Exosomes and hyaluronic acid influence tissue regeneration and may be used as an alternative to more conventional wound treatment methods. This study compared how well hyaluronic acid from the human umbilical cord and exosomes from fibroblast cells heal burn wounds in a preclinical model. METHODS: Ninety-six male Westar rats were used and allocated into four groups: The treatment group received 10% hyaluronic acid (HA); the treatment group received 300 l of exosome solution (EX); the treatment group received phenytoin (PC); the negative control group received no treatment (NC). The wound healing process was evaluated after 3, 6, 9, and 12 days. Histopathological analysis was done on the skin biopsy taken from the wounds. Re-epithelialization, inflammatory cells (PMNs), lymphocytes (LYMs), granulation tissue, collagen maturation (fibrosis), and eschar formation parameters were assessed for histopathological evaluation. On a scale from 0 to 4, each parameter received a score. RESULTS: Compared to the PC and NC groups, the median score for re-epithelialization was greater in the HA and EX groups (P < 0.05). At three days, PMN abundance distinguished the PC and NC groups from the HA and EX groups (P < 0.01). Compared to the PC and NC groups, the HA and EX groups had a lower median LYM score (P < 0.01). We found no statistical difference between the four groups for granulation tissue and fibrosis (P > 0.05). The EX group had a lower average score for eschar formation than the PC, NC, and HA groups (P < 0.01). The HA and EX groups demonstrated faster healing in the clinical and microscopic examinations than the NC and PC groups. CONCLUSION: The results showed that hyaluronic acid and exosomes improved wound healing. Also, the study demonstrated that hyaluronic acid has better effects in the re-epithelization. The exosome was more effective than HA in eschar formation. Both compounds were more influential in the PMNs and LYMs parameters than other groups. The combination of both compounds should be assessed further to achieve better therapeutic effects on wound healing.

Echocardiographic assessment of intravenous administration of medetomidine and xylazine hydrochloride at different sedative doses in one-humped camel calves (Camelus dromedarious).

Echocardiography illustrates a convenient and noninvasive tool for measuring cardiac output (CO) changes after administration of sedative drugs, but it is unknown in camelids practice. The aim of present study was to investigate echocardiographic effects of intravenous (IV) injection of medetomidine and xylazine in camel calves. Twenty apparently healthy immature male one-humped camel calves (Camelus dromedarious) were divided into four groups (five animals in each treatment). Medetomidine and xylazine were injected into the left jugular vein at two different doses of 10.00 and 20.00 µg kg-1 and 0.20 and 0.40 mg kg-1, respectively. Effects on some selected echocardiographic parameters were recorded at different intervals, before drug administrations (baseline) and after 3, 60 and 120 min. Data were analyzed by repeated measure, ANOVA test, then relevance and significance were taken as p ≤ 0.05. Significant decrease in fractional shortening percentage (FS%), ejection fraction percentage (EF%), stroke volume (SV), heart rate (HR) and subsequent CO were noticeable 3 min after drug administration in medetomidine high dose (MH), medetomidine low dose (ML) and xylazine high dose (XH) groups (p ≤ 0.05), furthermore at this time significant decrease in left ventricular mass (LVmass) and left ventricular systolic time intervals were seen in these groups, however, in xylazine low dose (XL) group, the lowest level of most echocardiographic parameters were detectable after 60 min. High dose IV injection of medetomidine was associated with significant decrease in most echocardiographic parameters without echocardiographic arrhythmia. Although, ML and XH groups had the same effects on echocardiographic indices but the intensity and duration were less than MH group.

Recombinant nanobody against MUC1 tandem repeats inhibits growth, invasion, metastasis, and vascularization of spontaneous mouse mammary tumors.

Alteration in glycosylation pattern of MUC1 mucin tandem repeats during carcinomas has been shown to negatively affect adhesive properties of malignant cells and enhance tumor invasiveness and metastasis. In addition, MUC1 overexpression is closely interrelated with angiogenesis, making it a great target for immunotherapy. Alongside, easier interaction of nanobodies (single-domain antibodies) with their antigens, compared to conventional antibodies, is usually associated with superior desirable results. Herein, we evaluated the preclinical efficacy of a recombinant nanobody against MUC1 tandem repeats in suppressing tumor growth, angiogenesis, invasion, and metastasis. Expressed nanobody demonstrated specificity only toward MUC1-overexpressing cancer cells and could internalize in cancer cell lines. The IC50 values (the concentration at which the nanobody exerted half of its maximal inhibitory effect) of the anti-MUC1 nanobody against MUC1-positive human cancer cell lines ranged from 1.2 to 14.3 nm. Similar concentrations could also effectively induce apoptosis in MUC1-positive cancer cells but not in normal cells or MUC1-negative human cancer cells. Immunohistochemical staining of spontaneously developed mouse breast tumors prior to in vivo studies confirmed cross-reactivity of nanobody with mouse MUC1 despite large structural dissimilarities between mouse and human MUC1 tandem repeats. In vivo, a dose of 3 µg nanobody per gram of body weight in tumor-bearing mice could attenuate tumor progression and suppress excessive circulating levels of IL-1a, IL-2, IL-10, IL-12, and IL-17A pro-inflammatory cytokines. Also, a significant decline in expression of Ki-67, MMP9, and VEGFR2 biomarkers, as well as vasculogenesis, was evident in immunohistochemically stained tumor sections of anti-MUC1 nanobody-treated mice. In conclusion, the anti-MUC1 tandem repeat nanobody of the present study could effectively overcome tumor growth, invasion, and metastasis.

Development and evaluation of methods for vertebral heart score determination in guinea pig (Cavia porcellus).

Cardiac problems are highly fetal diseases among exotic animals, not only in the rabbit which is prone to such diseases but also in the guinea pig (Cavia porcellus). In rodents, imaging studies such as thoracic radiography are more practical and easier to perform than echocardiography. Cardiac size is primarily evaluated using vertebral heart size (VHS) as reported in ferrets and rabbits. We therefore attempted to determine standard cardiac dimensions in the guinea pig by thoracic radiography using VHS. The purpose of this research was getting an indicator of the normal range of male and female, mature and apparently healthy guinea pigs heart. Standard radiographs of the thorax in lateral and ventro-dorsal (VD) views were taken and interpreted. In our study to determine VHS in VD view conventional method was used. In addition to conventional VHS method, two other measurement methods were performed in lateral view. Statistical analyses were performed with a SPSS Software and Mann-Whitney U test to compare results. Mean and standard deviation were also calculated. According to the results, the total average of VHS in lateral view by the first method was 7.80 ± 0.12, by the second method was 7.80 ± 0.16, by the third method was 7.60 ± 0.15 and the total of average of VHS in VD view was 9.20 ± 0.23. According to present research findings, gender had no meaningful effect on the measuring of the heart size. More researches on the same age and the same species guinea pigs are needed for more accurate evaluation.

Capture-free deactivation of CTCs in the bloodstream; a metastasis suppression method by electrostatic stimulation of the peripheral blood.

While limited investigations have been reported on CTC elimination and its profits, recently, some new works were reported on detection followed by the destruction of CTCs. Limitations and complications of CTC capturing procedures have highly reduced the chance of selective destruction of CTCs in the bloodstream in the therapeutic guidelines of the patients. Here, we selectively deactivated the invasive function of CTCs during their circulation in the bloodstream by exposing the whole blood to pure positive electrostatic charge stimulation (PPECS). Our treatment suppressed pulmonary metastasis and extended the survival of the mice had been intravenously injected by electrostatically deactivated 4T1 breast cancer CTCs. Moreover, the number of cancerous lung nodules was drastically reduced in the mice injected by treated CTCs in comparison with the non-treated cohort. Evaluating the side effect of the PPECS on the blood components revealed no major effect on the functional properties of the white blood cells, and just a negligible fraction (∼10%) was damaged during this process. This approach does not need any capturing or targeting of CTCs from the blood as it is focused on perturbing the electrical function of negatively-charged tumor cells after being exposed to positive electrostatic charges. Taken together, continuous in-vivo deactivation of CTCs by PPECS with no requirement to complicated capturing protocols may improve the survival of cancer patients.

Ultrasound-guided induced fetal death, an alternative method for induction of abortion in the bitch.

This study was performed to achieve abortion in a short time and with minimum side effects in a bitch, as an alternative method for termination of unwanted pregnancy. The experimentation was performed on 10 privately owned crossbred pregnant bitches, in their late second trimester of pregnancy, having a variable number of fetuses (3 to 9). Fetal death was induced by transabdominal intracardiac injection of potassium chloride (KCl) into the fetal heart under ultrasonographic guidance. Pregnancy was terminated within 36 to 72 hr (51.60 ± 16.04 hr) and none of the patients experienced any side effects or clinical complications. Data presented in this report provided evidence for the possible use of this technique to selectively reduce the number of canine fetuses without terminating the whole pregnancy. Ultrasound-guided induced fetal death is a safe procedure and a viable method for the induction of abortion in a short time and with no apparent side effects.

Nanoporous platinum needle for cancer tumor destruction by EChT and impedance-based intra-therapeutic monitoring.

Herein, we present a new design on the Single Needle Electrochemical Therapy (SNEChT) method by introducing some major improvements, including a nanoporous platinum electrode, tunable in situ anode size that depends on the width and location of the tumor, and the capability of measuring the efficacy of therapy based in intra-therapeutic impedance recording by the same EChT needle. It could have significant implications in optimizing EChT operative conditions. The nanoporous Pt electrode increased the interactive surface with a tumor, and produced a higher amount of current with lower stimulating DC voltage. The tunable anode size prevents the over-acidification of treated or non-desired lesions. Hence, this feature reduced the over distribution of tissue. Monitoring the impedance during the therapy clearly informs us about the local destruction of the tumor in each location. Thus, we can be informed about the threshold of tissue acidosis with the lowest electrical stimulation. The insertion of one needle with a tunable anode length for both precise therapy and impedance-based intra-therapeutic monitoring will shed new light on the applications of EChT.

Microneedle-Based Generation of Microbubbles in Cancer Tumors to Improve Ultrasound-Assisted Drug Delivery.

Production of local microbubbles (MBs) with dense distribution in tumor environment is achieved by developing an integrated electrochemical stimulator on a microfabricated silicon needle covered by zinc-oxide nanowires (ZnONWs). MBs are then exploded by external ultrasonic actuation, which induce microcavitations in tumor cells followed by direct entrance of anticancer drugs into cancer cells. This system, named ZnO nanowire-based microbubble generator probe (ZnONW-MGP), is tested on tumorized mice models (by MC4L2 breast cell lines). Mice treated by ZnONW-MGP have ≈82% reduction in tumor size within 10 days with just 25% of conventional dose of paclitaxel while in the absence of the system, they have just a 15% reduction in tumor size. Presence of ZnO nanostructures on microneedles strongly reduces the size of MBs and enhances the efficacy of the sonoporation.