A bioluminescent Pseudomonas aeruginosa wound model reveals increased mortality of type 1 diabetic mice to biofilm infection.

OBJECTIVE: To examine how bacterial biofilms, as contributing factors in the delayed closure of chronic wounds in patients with diabetes, affect the healing process. METHOD: We used daily microscopic imaging and the IVIS Spectrum in vivo imaging system to monitor biofilm infections of bioluminescent Pseudomonas aeruginosa and evaluate healing in non-diabetic and streptozotocin-induced diabetic mice. RESULTS: Our studies determined that diabetes alone did not affect the rate of healing of full-depth murine back wounds compared with non-diabetic mice. The application of mature biofilms to the wounds significantly decreased the rate of healing compared with non-infected wounds for both non-diabetic as well as diabetic mice. Diabetic mice were also more severely affected by biofilms displaying elevated pus production, higher mortality rates and statistically significant increase in wound depth, granulation/fibrosis and biofilm presence. Introduction of a mutant Pseudomonas aeruginosa capable of producing high concentrations of cyclic di-GMP did not result in increased persistence in either diabetic or non-diabetic animals compared with the wild type strain. CONCLUSION: Understanding the interplay between diabetes and biofilms may lead to novel treatments and better clinical management of chronic wounds.

Improved efficacy of a novel anti-angiogenic drug combination (TL-118) against colorectal-cancer liver metastases; MRI monitoring in mice.

BACKGROUND: The poor prognosis of patients with colorectal-cancer liver metastases (CRLM) and the insufficiency of available treatments have raised the need for alternative curative strategies. We aimed to assess the therapeutic potential of TL-118, a new anti-angiogenic drug combination, for CRLM treatment, in a mouse model. METHODS: The therapeutic potential of TL-118 was evaluated and compared with B20-4.1.1 (B20; anti-VEGF antibody) and rapamycin in CRLM-bearing mice. Tumour progression and the vascular changes were monitored by MRI. Additionally, mice survival, cell proliferation, apoptosis and vessel density were evaluated. RESULTS: This study demonstrated an unequivocal advantage to TL-118 therapy by significantly prolonging survival (threefold) and reducing metastasis perfusion and vessel density (ninefold). The underlying mechanism for TL-118-treatment success was associated with hepatic perfusion attenuation resulting from reduced nitric-oxide (NO) serum levels as elucidated by using hemodynamic response imaging (HRI, a functional MRI combined with hypercapnia and hyperoxia). Further, systemic hepatic perfusion reduction during the initial treatment phase by adding NO inhibitor has proven to be essential for reaching maximal therapeutic effects for both TL-118 and B20. CONCLUSION: TL-118 harbours a potential clinical benefit to CLRM patients. Moreover, the reduction of hepatic perfusion at early stages of anti-angiogenic therapies by adding NO inhibitor is crucial for achieving maximal anti-tumour effects.

Milk Contamination by Mycobacterium tuberculosis Complex, Implications for Public Health in Amazonas, Brazil.

ABSTRACT: In Brazil, contamination of raw milk with Mycobacterium tuberculosis complex (MTC) has been reported in several states. The highest rate of consumption of raw milk and its derivatives in Brazil occurs in Amazonas. This state also has the highest prevalence of tuberculosis in both humans and livestock. We assessed the contamination of cow's milk and buffalo's milk with MTC in Amazonas, focusing on Mycobacterium bovis, the species most commonly found in cattle and buffalo. In 2019, 250 samples of raw milk (91 from cattle, 159 from buffalo) were collected before processing from three milk plants in the state of Amazonas. The samples were placed into 21 pools and analyzed using shotgun metagenomic sequencing and taxonomic classification with Kraken 2 and MegaBLAST. To confirm the identity of mycobacterial species found, BLASTN was used to identify specific genomic positions in the TbD1 and RD1 regions and flanking RD4 region. MTC genetic material was identified in all pools of raw milk. Genetic material consistent with M. bovis was identified in seven pools of raw milk (1 from cattle, 6 from buffalo). Buffalo's milk had significantly higher MTC reads than did cow's milk. The common practice of consumption of raw milk and its derivatives in Amazonas presents a risk to public health. Urgent measures to prevent transmission of foodborne tuberculosis are needed in the Amazon region. Greater efforts and resources also should be directed toward elimination of bovine tuberculosis in cattle and buffalo herds in Amazonas and the rest of Brazil.

Counteracting age-related VEGF signaling insufficiency promotes healthy aging and extends life span.

Aging is an established risk factor for vascular diseases, but vascular aging itself may contribute to the progressive deterioration of organ function. Here, we show in aged mice that vascular endothelial growth factor (VEGF) signaling insufficiency, which is caused by increased production of decoy receptors, may drive physiological aging across multiple organ systems. Increasing VEGF signaling prevented age-associated capillary loss, improved organ perfusion and function, and extended life span. Healthier aging was evidenced by favorable metabolism and body composition and amelioration of aging-associated pathologies including hepatic steatosis, sarcopenia, osteoporosis, "inflammaging" (age-related multiorgan chronic inflammation), and increased tumor burden. These results indicate that VEGF signaling insufficiency affects organ aging in mice and suggest that modulating this pathway may result in increased mammalian life span and improved overall health.

Cyclosporin a induces renal episodic hypoxia.

AIM: Cyclosporin A (CsA) causes renal toxicity. The underlying mechanisms are incompletely understood, but may involve renal hypoxia and hypoxia-inducible factors (Hifs). We sought for hypoxia and Hif in mouse kidneys with CsA-induced toxicity, assessed their time course, Hif-mediated responses and the impact of interventional Hif upregulation. METHODS: Mice received CsA or its solvent cremophore for up to 6 weeks. Low salt diet (Na+ ↓) was given in combination with CsA to enhance toxicity. We assessed fine morphology, renal function, blood oxygen level-dependent magnetic resonance imaging under room air and following changes in breathing gas composition which correlate with vascular reactivity, pimonidazole adducts (which indicate O2 tensions below 10 mmHg), Hif-α proteins, as well as expression of Hif target genes. Stable Hif upregulation was achieved by inducible, Pax8-rtTA-based knockout of von Hippel-Lindau protein (Vhl-KO), which is crucial for Hif-α degradation. RESULTS: Cyclosporin A transiently increased renal deoxyhaemoglobin (R2*). Augmented vascular reactivity was observed at 2 h, but decreased at 24 h after CsA treatment. Na+ ↓/CsA provoked chronic renal failure with tubular degeneration and interstitial fibrosis. Nephron segments at risk for injury accumulated pimonidazole adducts, as well as Hif-α proteins. Remarkably, Hif target gene expression remained unchanged, while factor-inhibiting Hif (Fih) was enhanced. Na+ ↓/CsA/Vhl-KO aggravated morpho-functional outcome of chronic renal CsA toxicity. CONCLUSIONS: Cyclosporin A provokes episodic hypoxia in nephron segments most susceptible to chronic CsA toxicity. Fih is upregulated and likely blocks further Hif activity. Continuous tubular Hif upregulation via Vhl-KO worsens the outcome of chronic CsA-induced renal toxicity.

Neovascularization induced growth of implanted C6 glioma multicellular spheroids: magnetic resonance microimaging.

Magnetic resonance imaging has been used to follow noninvasively tumor neovascularization and tumor growth in a model system of multicellular C6 rat glioma spheroids implanted s.c. in nude mice. By positioning a single spheroid approximately 1 cm from the site of incision both the vascularization of the tumor and the wound healing processes were spatially separated and could be simultaneously followed. The model proposed here provides defined initial conditions of tumor geometry and cell proliferative status and separation of initial tumor growth from neovascularization. Magnetic susceptibility relaxation provided an intrinsic marker for blood containing vessels. The implanted spheroid induced vessel growth within 4 days after implantation that was geometrically oriented toward the spheroid and distinct from wound healing at the site of incision. Volume measurements showed a corresponding 4-day lag in growth followed by Gompertz progression. Sham implantation of agarose beads of similar diameter showed no induction of vessel growth, ruling out a direct effect of wound healing. The new vessels penetrating the tumor were highly permeable to the contrast reagent gadolinium-diethylenetriaminepentaacetic acid. This permeability may be due to the action of vascular endothelial growth factor, a major angiogenic growth factor in this system, and a potent permeability factor.

Cyclocreatine accumulation leads to cellular swelling in C6 glioma multicellular spheroids: diffusion and one-dimensional chemical shift nuclear magnetic resonance microscopy.

Cyclocreatine, an analogue of creatine, inhibits tumor cell proliferation in vitro and in vivo. The effects of cyclocreatine in large C6 glioma multicellular spheroids were mapped here by magnetic resonance microscopy. Diffusion-weighted images of C6 glioma spheroids resolved the bright viable rim and the dark necrotic center. Sequential sets of diffusion images, following cyclocreatine administration, showed increasing self-diffusion coefficients of the intracellular water in the viable rim (0.49 x 10(-5) cm2/s for untreated spheroids, 0.62 x 10(-5) cm2/s after 48 h perfusion with 20 mM cyclocreatine). This fact correlated with cellular swelling apparent in histological sections. The radial distribution of cyclocreatine and soluble lipids across perfused C6 spheroids was measured by one-dimensional chemical shift imaging. Cyclocreatine accumulation was prominent throughout the viable cell layer, with no cyclocreatine accumulation in the necrotic center. In both cyclocreatine-treated and control spheroids the lipid signal was highest in the necrotic center and lower in the inner viable cell layer.

In vivo prediction of vascular susceptibility to vascular susceptibility endothelial growth factor withdrawal: magnetic resonance imaging of C6 rat glioma in nude mice.

One of the hallmarks of tumor neovasculature is the prevalence of immature vessels manifested by the low degree of recruitment of vascular mural cells such as pericytes and smooth muscle cells. This difference in the architecture of the vascular bed provides an important therapeutic window for inflicting tumor-selective vascular damage. Here we demonstrate the application of gradient echo magnetic resonance imaging (MRI) for noninvasive in vivo mapping of vascular maturation, manifested by the ability of mature vessels to dilate in response to elevated levels of CO2. Histological alpha-actin staining showed a match between dilating vessels detected by MRI and vessels coated with smooth muscle cells. Switchable, vascular endothelial growth factor (VEGF)-overexpressing tumors (C6-pTET-VEGF rat glioma s.c. tumors in nude mice) displayed high vascular function and significant vascular damage upon VEGF withdrawal. However, damage was restricted to nondilating vessels, whereas mature dilating tumor vessels were resistant to VEGF withdrawal. Thus, MRI provides in vivo visualization of vascular maturity and prognosis of vascular obliteration induced by VEGF withdrawal.

In vivo molecular imaging of met tyrosine kinase growth factor receptor activity in normal organs and breast tumors.

Molecular imaging techniques allow visualization of specific gene products and their physiological processes in living tissues. In this study, we present a new approach for molecular imaging of endogenous tyrosine kinase receptor activity. Met and its ligand hepatocyte growth factor scatter factor (HGF/SF), which mediate mitogenicity, tumorigenicity, and angiogenesis, were used as a model. HGF/SF and Met play a significant role in the pathogenesis and biology of a wide variety of human epithelial cancers and, therefore, may serve as potential targets for cancer prognosis and therapy. We have shown previously that in vitro activation of Met by HGF/SF increases oxygen consumption. In this study, we demonstrate that Met activation in vivo by HGF/SF alters the hemodynamics of normal and malignant Met-expressing tissues. Tumor-bearing BALB/C mice were i.v. injected with HGF/SF and imaged using magnetic resonance imaging (MRI) and Doppler ultrasound. Organs and tumors expressing high levels of Met showed the most substantial alteration in blood oxygenation levels as measured by blood oxygenation level depended (BOLD)-MRI. No significant alteration was observed in tumors or organs that does not express Met. In the liver, which expresses high levels of Met, MRI signal alteration of about 60% was observed. In the kidneys, signal alteration was approximately 30%, and no change was observed in muscles. The extent of MRI signal alteration was also in correlation with HGF/SF doses. Injection of 7 and 170 ng/g body weight resulted in signal alteration of 5% and 30%, respectively, in tumors. Doppler ultrasound measurements demonstrated that these MRI changes are at least partially attributable to altered blood flow. These hemodynamic alterations, measured by MRI and Doppler ultrasound, were used in this study for the molecular imaging of Met activity in vivo. This novel molecular imaging technique may be used for in vivo diagnosis, prognosis, and therapy of Met-expressing tumors.

The antiangiogenic agent linomide inhibits the growth rate of von Hippel-Lindau paraganglioma xenografts to mice.

The aim of this study was to ascertain the potential usefulness of the antiangiogenic compound linomide for treatment of von Hippel-Lindau (VHL)-related tumors. Paraganglioma tissue fragments obtained at surgery from a VHL type 2a patient were transplanted s.c. to male BALB/c nu/nu (nude) mice: (a) 2-3-mm fragments for "prevention" experiments; and (b) 2-3-mm fragments allowed to grow to 1 cm for "intervention" studies. Both groups received either 0.5 mg/ml linomide in drinking water or acidified water and were followed until tumor diameter reached 3 cm or for 4 weeks. In both the prevention and intervention experiments, a significant diminution of tumor size and weight was observed in the drug-treated animals. In vivo nuclear magnetic resonance analysis of tumor blood flow in linomide-treated animals showed localization of blood vessels almost exclusively to the periphery of the poorly vascularized tumors with a significant reduction of both vascular functionality and vasodilation. Histological examination of tumors from linomide-treated animals revealed marked avascularity. Treated animals also displayed a 2.4-fold reduction of tumor vascular endothelial growth factor mRNA levels. Taken together, our data indicate that in VHL disease, therapy directed at inhibition of constitutively expressed VEGF induction of angiogenesis by VHL tumors may constitute an effective medical treatment.

Inhibition of neovascularization and tumor growth, and facilitation of wound repair, by halofuginone, an inhibitor of collagen type I synthesis.

Halofuginone, an inhibitor of collagen alpha1(I) gene expression was used for the treatment of subcutaneously implanted C6 glioma tumors. Halofuginone had no effect on the growth of C6 glioma spheroids in vitro, and these spheroids showed no collagen alpha1(I) expression and no collagen synthesis. However, a significant attenuation of tumor growth was observed in vivo, for spheroids implanted in CD-1 nude mice which were treated by oral or intraperitoneal (4 microg every 48 hours) administration of halofuginone. In these mice, treatment was associated with a dose-dependent reduction in collagen alpha1(I) expression and dose- and time-dependent inhibition of angiogenesis, as measured by MRI. Moreover, halofuginone treatment was associated with improved re-epithelialization of the chronic wounds that are associated with this experimental model. Oral administration of halofuginone was effective also in intervention in tumor growth, and here, too, the treatment was associated with reduced angiogenic activity and vessel regression. These results demonstrate the important role of collagen type I in tumor angiogenesis and tumor growth and implicate its role in chronic wounds. Inhibition of the expression of collagen type I provides an attractive new target for cancer therapy.

Intercellular communication between vascular smooth muscle and endothelial cells mediated by heparin-binding epidermal growth factor-like growth factor and vascular endothelial growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen and migration factor for vascular smooth muscle cells (SMC), promoted neovascularization in vivo in the rabbit cornea. MRI demonstrated quantitatively the angiogenic effect of HB-EGF when introduced subcutaneously into nude mice. HB-EGF is not directly mitogenic to endothelial cells but it induced the migration of bovine endothelial cells and release of endothelial cell mitogenic activity from bovine vascular SMC. This mitogenic activity was specifically blocked by neutralizing anti-vascular endothelial growth factor (VEGF) antibodies. In contrast, EGF or transforming growth factor-alpha (TGF-alpha) had almost no effect on release of endothelial mitogenicity from SMC. In addition, RT-PCR analysis demonstrated that VEGF165 mRNA levels were increased in vascular SMC 4-10-fold by 0.35-2 nM of HB-EGF, respectively. Our data suggest that HB-EGF, as a mediator of intercellular communication, may play a new important role in supporting wound healing, tumor progression and atherosclerosis by stimulating angiogenesis.

Informational needs of parents of sick neonates.

Sixty-one parents of 43 neonates in a neonatal intensive care unit were interviewed within 3 days of their first conference with the neonatologist to assess their needs for medical information. The conference with the physician and the interview with the investigator were audiotaped. Information given by the physician and information recalled by the parents was categorized and rated by the investigator. The physicians gave very detailed information about diagnosis in 77% of cases whereas 39% of the parents recalled diagnostic information in great detail. Respective percentages for prognosis were 16 and 33; for current management (eg, investigation, therapy), 28 and 66; and for cause of illness, 16 and 18. The statistical significance of the differences between the very detailed information int he physician-parent conferences and in the parent-investigator interviews was, by category, less than .002, less than .041, less than .004, and not significant, respectively. Information in the respective categories was described as "most helpful" by 20%, 67%, 90%, and 8% of parents. All but one of the parents stated that they wanted the whole truth. Physician uncertainty in providing information was significantly associated with severity of illness. It is concluded that while parents wanted information in all categories, they paid most attention to that relating to management. Physician-parent discordances with respect to information about management, diagnosis, and prognosis suggest directions for future investigation of the optimal content of information for parents in this context.

Preliminary report on informed consent for mental capacity assessments.

The goal of the study was to investigate the issue of informed consent for mental capacity. Seventeen clients referred to the Competency Clinic at the Baycrest Centre for Geriatric Care were interviewed after the assessment had been completed. Their responses to questions regarding the reasons for and possible consequences of the assessment indicated a range of understanding and of capacity to give informed consent. The results are discussed in terms of the appropriateness of having a fairly low threshold for informed consent in situations where other capacities are already in question.

MRI multiparametric hemodynamic characterization of the normal brain.

Characterization of the brain's vascular system is of major clinical importance in the assessment of patients with cerebrovascular disease. The aim of this study was to characterize brain hemodynamics using multiparametric methods and to obtain reference values from the healthy brain. A multimodal magnetic resonance imaging (MRI) study was performed in twenty healthy subjects, including dynamic susceptibility contrast imaging and blood oxygen level dependence (BOLD) during hypercapnia and carbogen challenges. Brain tissues were defined using unsupervised cluster analysis based on these three methods, and several hemodynamic parameters were calculated for gray matter (GM), white matter (WM), blood vessels and dura (BVD); the three main vascular territories within the GM; and arteries and veins defined within the BVD cluster. The carbogen challenge produced a BOLD signal twice as high as the hypercapnia challenge, in all brain tissues. The three brain tissues differed significantly from each other in their hemodynamic characteristics, supporting the graded vascularity of the tissues, with BVD>GM>WM. Within the GM cluster, a significant delay of ∼1.2 s of the bolus arrival time was detected within the posterior cerebral artery territory relative to the middle and anterior cerebral artery territories. No differences were detected between right and left middle cerebral artery territories for all hemodynamic parameters. Within the BVD cluster, a significant delay of ∼1.9 s of the bolus arrival time was detected within the veins relative to the arteries. This parameter enabled to differentiate between the various blood vessels, including arteries, veins and choroid plexus. This study provides reference values for several hemodynamic parameters, obtained from healthy brains, and may be clinically important in the assessment of patients with various vascular pathologies.

Classification of suspected liver metastases using fMRI images: a machine learning approach.

This paper presents a machine-learning approach to the interactive classification of suspected liver metastases in fMRI images. The method uses fMRI-based statistical modeling to characterize colorectal hepatic metastases and follow their early hemodynamical changes. Changes in hepatic hemodynamics are evaluated from T2*-W fMRI images acquired during the breathing of air, air-CO2, and carbogen. A classification model is build to differentiate between tumors and healthy liver tissues. To validate our method, a model was built from 29 mice datasets, and used to classify suspicious regions in 16 new datasets of healthy subjects or subjects with metastases in earlier growth phases. Our experimental results on mice yielded an accuracy of 78% with high precision (88%). This suggests that the method can provide a useful aid for early detection of liver metastases.

Children's understanding of the risks and benefits associated with research.

OBJECTIVE: The objective of the current study was to maximise the amount of information children and adolescents understand about the risks and benefits associated with participation in a biomedical research study. DESIGN: Participants were presented with one of six hypothetical research protocols describing how to fix a fractured thigh using either a "standard" cast or "new" pins procedure. Risks and benefits associated with each of the treatment options were manipulated so that for each one of the six protocols there was either a correct or ambiguous choice. PARTICIPANTS AND SETTING: Two hundred and fifty one children, ages 6-15 (53% boys), and 237 adults (30% men) were interviewed while waiting for a clinic appointment at the Hospital for Sick Children. RESULTS: Using standardised procedures and questionnaires, it was determined that most participants, regardless of age group, were able to understand the basic purpose and procedures involved in the research, and most were able to choose the "correct" operation. The younger children, however, showed an overall preference for a cast operation, whereas the older participants were more likely to choose the pins. CONCLUSIONS: By creating age appropriate modules of information, children as young as six years can understand potentially difficult and complex concepts such as the risks and benefits associated with participation in biomedical research. It appears, however, that different criteria were used for treatment preference, regardless of associated risks; older participants tended to opt for mobility (the pins procedure) whereas younger participants stayed with the more familiar cast operation.

Analysis of subcutaneous angiogenesis by gradient echo magnetic resonance imaging.

The goal of this study was to develop an experimental method for noninvasive analysis of angiogenesis, namely the sprouting of capillaries from existing blood vessels. Angiogenesis was assayed in the subcutaneous vasculature of nude mice in a region of 3 x 3 cm that included in its center a defined angiogenic stimulus. Angiogenic stimuli included agarose beads containing angiogenic growth factors, multicellular tumor spheroids, and dermal incisions. Highly significant correlation (r = 0.905, P = 0.0001) was found between the apparent vessel density determined by gradient echo MRI and the density of blood-containing vessels determined postmortem. The functionality of the neovasculature was demonstrated in mice breathing alternatingly carbogen or 95% air/5% CO2. Large signal enhancement with carbogen breathing corresponded to regions of high vessel density. The assay reported here can be applied for the study of dermal wound healing, primary vascularization of subcutaneous implants, and for measuring the activity of angiogenic and antiangiogenic agents.

The influence of down's syndrome on sibling interaction.

Home observations were done on sibling interactions in 31 families with a child having Down's syndrome and a non-handicapped sibling. The siblings with Down's syndrome initiated less prosocial and agonistic behaviour, but imitated more frequently than their non-handicapped siblings. These effects were found regardless of birth order. There were no effects of gender. Higher levels of prosocial behaviours among large interval dyads and in dyads with a second-born Down's syndrome child were primarily due to the age of the non-handicapped sibling. Results were similar to those in previous "normative" studies of sibling interactions.

Regulation of angiogenesis by hypoxic stress: from solid tumours to the ovarian follicle.

The preovulatory follicle provides a unique physiological example of rapid growth accompanied by neovascularization, two processes that are generally characteristic of pathologies such as wound repair or malignancy. During the hours preceding ovulation, follicular growth is accompanied by elevated levels of messenger RNA for vascular endothelial growth factor (VEGF). Angiogenic activity, mediated by VEGF, is manifested in the peripheral blood vessels surrounding the follicle, that show capillary sprouting and increased vascular permeability. Following ovulation, rapid infiltration of capillaries through the follicular wall is essential for the formation of the corpus luteum. In this review we compare the preovulatory follicle with a popular model of avascular solid tumour growth, namely the multicellular tumour spheroid, in particular the role of hypoxic stress in the regulation of angiogenesis in both systems.