p66ShcA promotes malignant breast cancer phenotypes by alleviating energetic and oxidative stress.

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.

Comparative Study of Histotripsy Pulse Parameters Used to Inactivate Escherichia coli in Suspension.

OBJECTIVE: Bacterial loads can be effectively reduced using cavitation-mediated focused ultrasound, or histotripsy. In this study, gram-negative bacteria (Escherichia coli) in suspension were used as model bacteria to evaluate the effectiveness of two regimens of histotripsy treatments: cavitation histotripsy (CH) and boiling histotripsy (BH). METHODS: Ten-milliliter volumes of Escherichia coli were treated at different negative focal pressure amplitudes and over time periods up to 40 min. Cavitation activity was characterized with coaxial passive cavitation detection (PCD) and synchronized plane wave B-mode imaging. RESULTS: CH treatments exhibited a threshold behavior that was consistent with PCD metrics of cavitation. Above the threshold, bacterial inactivation followed a monotonically increasing log-linear relationship that indicated an exponential inactivation rate. BH exhibited no threshold, but instead followed a different monotonically increasing inactivation rate. Inactivation rates were larger for BH at or below the CH threshold, and larger for CH substantially above the threshold. CH studies performed at different pulse lengths at the same duty cycle had similar inactivation rates, suggesting that at any given pressure amplitude, the "on time" was the most important variable for inactivating E. coli. The maximum inactivation was produced by CH at the highest pressure amplitudes used, leading to a log reduction >4.2 for a 40 min treatment. CONCLUSION: The results of this study suggest that both CH and BH can be used to inactivate E. coli in suspension, with the optimal regimen depending on the attainable peak negative focal pressure at the target.

Chronic effects of pulsed high intensity focused ultrasound aided delivery of gemcitabine in a mouse model of pancreatic cancer.

Pulsed high intensity focused ultrasound (pHIFU) is a non-invasive method that allows to permeabilize pancreatic tumors through inertial cavitation and thereby increase the concentration of systemically administered drug. In this study the tolerability of weekly pHIFU-aided administrations of gemcitabine (gem) and their influence on tumor progression and immune microenvironment were investigated in genetically engineered KrasLSL.G12D/þ; p53R172H/þ; PdxCretg/þ (KPC) mouse model of spontaneously occurring pancreatic tumors. KPC mice were enrolled in the study when the tumor size reached 4-6 mm and treated once a week with either ultrasound-guided pHIFU (1.5 MHz transducer, 1 ms pulses, 1% duty cycle, peak negative pressure 16.5 MPa) followed by administration of gem (n = 9), gem only (n = 5) or no treatment (n = 8). Tumor progression was followed by ultrasound imaging until the study endpoint (tumor size reaching 1 cm), whereupon the excised tumors were analyzed by histology, immunohistochemistry (IHC) and gene expression profiling (Nanostring PanCancer Immune Profiling panel). The pHIFU + gem treatments were well tolerated; the pHIFU-treated region of the tumor turned hypoechoic immediately following treatment in all mice, and this effect persisted throughout the observation period (2-5 weeks) and corresponded to areas of cell death, according to histology and IHC. Enhanced labeling by Granzyme-B was observed within and adjacent to the pHIFU treated area, but not in the non-treated tumor tissue; no difference in CD8 + staining was observed between the treatment groups. Gene expression analysis showed that the pHIFU + gem combination treatment lead to significant downregulation of 162 genes related to immunosuppression, tumorigenesis, and chemoresistance vs gem only treatment.

In Vivo Aberration Correction for Transcutaneous HIFU Therapy Using a Multielement Array.

One of the challenges of transcutaneous high-intensity focused ultrasound (HIFU) therapies, especially ones relying heavily on shock formation, such as boiling histotripsy (BH), is the loss of focusing from aberration induced by the heterogeneities of the body wall. Here, a methodology to execute aberration correction in vivo is proposed. A custom BH system consisting of a 1.5-MHz phased array of 256 elements connected to a Verasonics V1 system is used in pulse/echo mode on a porcine model under general anesthesia. Estimation of the time shifts needed to correct for aberration in the liver and kidney is done by maximizing the value of the coherence factor on the acquired backscattered signals. As this process requires multiple pulse/echo sequences on a moving target to converge to a solution, tracking is also implemented to ensure that the same target is used between each iteration. The method was validated by comparing the acoustic power needed to generate a boiling bubble at one target with aberration correction and at another target within a 5-mm radius without aberration correction. Results show that the aberration correction effectively lowers the acoustic power required to reach boiling by up to 45%, confirming that it indeed restored formation of the nonlinear shock front at the focus.

Non-Invasive Monitoring of Increased Fibrotic Tissue and Hyaluronan Deposition in the Tumor Microenvironment in the Advanced Stages of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinomas are characterized by a complex and robust tumor microenvironment (TME) consisting of fibrotic tissue, excessive levels of hyaluronan (HA), and immune cells. We utilized quantitative multi-parametric magnetic resonance imaging (mp-MRI) methods at 14 Tesla in a genetically engineered KPC (KrasLSL-G12D/+, Trp53LSL-R172H/+, Cre) mouse model to assess the complex TME in advanced stages of tumor development. The whole tumor, excluding cystic areas, was selected as the region of interest for data analysis and subsequent statistical analysis. Pearson correlation was used for statistical inference. There was a significant correlation between tumor volume and T2 (r = -0.66), magnetization transfer ratio (MTR) (r = 0.60), apparent diffusion coefficient (ADC) (r = 0.48), and Glycosaminoglycan-chemical exchange saturation transfer (GagCEST) (r = 0.51). A subset of mice was randomly selected for histological analysis. There were positive correlations between tumor volume and fibrosis (0.92), and HA (r = 0.76); GagCEST and HA (r = 0.81); and MTR and CD31 (r = 0.48). We found a negative correlation between ADC low-b (perfusion) and Ki67 (r = -0.82). Strong correlations between mp-MRI and histology results suggest that mp-MRI can be used as a non-invasive tool to monitor the tumor microenvironment.

Safety of Image-Guided Treatment of the Liver with Ultrasound and Microbubbles in an in Vivo Porcine Model.

Ultrasound and microbubbles are useful for both diagnostic imaging and targeted drug delivery, making them ideal conduits for theranostic interventions. Recent reports have indicated the preclinical success of microbubble cavitation for enhancement of chemotherapy in abdominal tumors; however, there have been limited studies and variable efficacy in clinical implementation of this technique. This is likely because in contrast to the high pressures and long cycle lengths seen in successful preclinical work, current clinical implementation of microbubble cavitation for drug delivery generally involves low acoustic pressures and short cycle lengths to fit within clinical guidelines. To translate the preclinical parameter space to clinical adoption, a relevant safety study in a healthy large animal is required. Therefore, the purpose of this work was to evaluate the safety of ultrasound cavitation treatment (USCTx) in a healthy porcine model using a modified Philips EPIQ with S5-1 as the focused source. We performed USCTx on eight healthy pigs and monitored health over the course of 1 wk. We then performed an acute study of USCTx to evaluate immediate tissue damage. Contrast-enhanced ultrasound exams were performed before and after each treatment to investigate perfusion changes within the treated areas, and blood and urine were evaluated for liver damage biomarkers. We illustrate, through quantitative analysis of contrast-enhanced ultrasound data, blood and urine analyses and histology, that this technique and the parameter space considered are safe within the time frame evaluated. With its safety confirmed using a clinical-grade ultrasound scanner and contrast agent, USCTx could be easily translated into clinical trials for improvement of chemotherapy delivery. This represents the first safety study assessing the bio-effects of microbubble cavitation from relevant ultrasound parameters in a large animal model.

STAT1 potentiates oxidative stress revealing a targetable vulnerability that increases phenformin efficacy in breast cancer.

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

Factors Affecting Tissue Cavitation during Burst Wave Lithotripsy.

Burst wave lithotripsy (BWL) is a technology under clinical investigation for non-invasive fragmentation of urinary stones. Under certain ranges of ultrasound exposure parameters, this technology can cause cavitation in tissue leading to renal injury. This study sought to measure the focal pressure amplitude needed to cause cavitation in vivo and determine its consistency in native tissue, in an implanted stone model and under different exposure parameters. The kidneys of eight pigs were exposed to transcutaneous BWL ultrasound pulses. In each kidney, two locations were targeted: the renal sinus and the kidney parenchyma. Each was exposed for 5 min at a set pressure level and parameters, and cavitation was detected using an active cavitation imaging method based on power Doppler ultrasound. The threshold was determined by incrementing the pressure amplitude up or down after each 5-min interval until cavitation occurred/subsided. The pressure thresholds were remeasured postsurgery, targeting an implanted stone or collecting space (in sham). The presence of a stone or sham surgery did not significantly impact the threshold for tissue cavitation. Targeting parenchyma instead of kidney collecting space and lowering the ultrasound pulse repetition frequency both resulted in an increased pressure threshold for cavitation.

HIV in Canada-surveillance report, 2019.

BACKGROUND: Human immunodeficiency virus (HIV) is a global public health issue. HIV has been nationally notifiable in Canada since 1985. The Public Health Agency of Canada (PHAC) monitors trends in new HIV diagnoses. OBJECTIVES: The objective of this surveillance report is to provide an overview of the epidemiology of reported HIV cases in 2019 in Canada. The report highlights 10-year trends (2010-2019). Data on HIV diagnosed through Immigration Medical Exams (IME) and trends in perinatal transmission of HIV are also presented. METHODS: PHAC monitors HIV through the HIV/AIDS Surveillance System, a passive, case-based system that collates non-nominal data submitted voluntarily by all Canadian provinces and territories. Descriptive analyses were conducted on national data. IME data were obtained from Immigration, Refugees and Citizenship Canada (IRCC), and data on HIV-exposed pregnancies were obtained through the Canadian Perinatal HIV Surveillance Program. RESULTS: In 2019, a total of 2,122 HIV diagnoses were reported in Canada (5.6 per 100,000 population). Saskatchewan reported the highest provincial diagnosis rate at 16.9 per 100,000 population. The 30 to 39-year age group had the highest HIV diagnosis rate at 12.7 per 100,000 population. While the rates for both males and females fluctuated in the past decade, since 2010 the rates among males decreased overall, while the rate among females increased slightly. As in previous years, the diagnosis rate for males in 2019 was higher than that for females (7.9 versus 3.4 per 100,000 population, respectively). The highest proportion of all reported adult cases with known exposure were gay, bisexual and other men who have sex with men (gbMSM, 39.7%), followed by cases attributed to heterosexual contact (28.3%) and among people who inject drugs (PWID, 21.5%). The number of migrants who tested positive for HIV during an IME conducted in Canada was 626. The one documented perinatal HIV transmission related to a mother who had not received antepartum or intrapartum antiretroviral therapy prophylaxis. CONCLUSION: The number and rate of reported HIV cases in Canada has remained relatively stable over the last decade, with minor year-to-year variations. As in previous years, the gbMSM and PWID populations represent a high proportion of HIV diagnoses, although a sizable number of cases were attributed to heterosexual contact. It is important to routinely monitor trends in HIV in light of pan-Canadian commitments to reduce the health impact of sexually transmitted and blood-borne infections by 2030.

p66ShcA potentiates the cytotoxic response of triple-negative breast cancers to PARP inhibitors.

Triple-negative breast cancers (TNBCs) lack effective targeted therapies, and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohort of patients do respond to PARPi in combination with chemotherapies. Moreover, recent studies suggest that an increase in levels of ROS may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that, in response to therapy-induced stress, p66ShcA stimulated ROS production, which, in turn, potentiated the synergy of PARPi in combination with doxorubicin in TNBCs. This p66ShcA-induced sensitivity relied on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin.

Magnetic resonance imaging biomarkers for pulsed focused ultrasound treatment of pancreatic ductal adenocarcinoma.

BACKGROUND: The robust fibroinflammatory stroma characteristic of pancreatic ductal adenocarcinoma (PDA) impedes effective drug delivery. Pulsed focused ultrasound (pFUS) can disrupt this stroma and has improved survival in an early clinical trial. Non-invasive methods to characterize pFUS treatment effects are desirable for advancement of this promising treatment modality in larger clinical trials. AIM: To identify promising, non-invasive pre-clinical imaging methods to characterize acute pFUS treatment effects for in vivo models of PDA. METHODS: We utilized quantitative magnetic resonance imaging methods at 14 tesla in three mouse models of PDA (subcutaneous, orthotopic and transgenic - KrasLSL-G12D/+ , Trp53LSL-R172H/+ , Cre or "KPC") to assess immediate tumor response to pFUS treatment (VIFU 2000 Alpinion Medical Systems; 475 W peak electric power, 1 ms pulse duration, 1 Hz, duty cycle 0.1%) vs sham therapy, and correlated our results with histochemical data. These pFUS treatment parameters were previously shown to enhance tumor permeability to chemotherapeutics. T1 and T2 relaxation maps, high (126, 180, 234, 340, 549) vs low (7, 47, 81) b-value apparent diffusion coefficient (ADC) maps, magnetization transfer ratio (MTR) maps, and chemical exchange saturation transfer (CEST) maps for the amide proton spectrum (3.5 parts per million or "ppm") and the glycosaminoglycan spectrum (0.5-1.5 ppm) were generated and analyzed pre-treatment, and immediately post-treatment, using ImageJ. Animals were sacrificed immediately following post-treatment imaging. The whole-tumor was selected as the region of interest for data analysis and subsequent statistical analysis. T-tests and Pearson correlation were used for statistical inference. RESULTS: Mean high-b value ADC measurements increased significantly with pFUS treatment for all models. Mean glycosaminoglycan CEST and T2 measurements decreased significantly post-treatment for the KPC group. Mean MTR and amide CEST values increased significantly for the KPC group. Hyaluronic acid focal intensities in the treated regions were significantly lower following pFUS treatment for all animal models. The magnetic resonance imaging changes observed acutely following pFUS therapy likely reflect: (1) Sequelae of variable degrees of microcapillary hemorrhage (T1, MTR and amide CEST); (2) Lower PDA glycosaminoglycan content and associated water content (glycosaminoglycan CEST, T2 and hyaluronic acid focal intensity); and (3) Improved tumor diffusivity (ADC) post pFUS treatment. CONCLUSION: T2, glycosaminoglycan CEST, and ADC maps may provide reliable quantitation of acute pFUS treatment effects for patients with PDA.

The Shc1 adaptor simultaneously balances Stat1 and Stat3 activity to promote breast cancer immune suppression.

Tyrosine kinase signalling within cancer cells is central to the establishment of an immunosuppressive microenvironment. Although tyrosine kinase inhibitors act, in part, to augment adaptive immunity, the increased heterogeneity and functional redundancy of the tyrosine kinome is a hurdle to achieving durable responses to immunotherapies. We previously identified the Shc1 (ShcA) scaffold, a central regulator of tyrosine kinase signalling, as essential for promoting breast cancer immune suppression. Herein we show that the ShcA pathway simultaneously activates STAT3 immunosuppressive signals and impairs STAT1-driven immune surveillance in breast cancer cells. Impaired Y239/Y240-ShcA phosphorylation selectively reduces STAT3 activation in breast tumours, profoundly sensitizing them to immune checkpoint inhibitors and tumour vaccines. Finally, the ability of diminished tyrosine kinase signalling to initiate STAT1-driven immune surveillance can be overcome by compensatory STAT3 hyperactivation in breast tumours. Our data indicate that inhibition of pY239/240-ShcA-dependent STAT3 signalling may represent an attractive therapeutic strategy to sensitize breast tumours to multiple immunotherapies.

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD.

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280-320-kDa oligomeric structure consisting of ∼6-7 subunits.

Microtubule-associated protein 2 and synaptophysin in the preterm and near-term ovine fetal brain and the effect of intermittent umbilical cord occlusion.

We have determined the change in immunoreactivity (IR) for microtubule-associated protein 2 (MAP-2) and synaptophysin (SYN) as markers for dendritic and presynaptic nerve development, respectively, in the ovine fetal brain with advancing gestation and in response to intermittent umbilical cord occlusion (UCO), which might then contribute to adverse neurodevelopment. Fetal sheep (control and experimental groups preterm at 111-115 and near term at 132-138 days of gestation; term = 145 days) were studied over 4 days with UCOs performed by inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then euthanized and fetal brains assessed for IR of MAP-2 and SYN. In control animals, the IR of SYN increased in the gray matter with advancing gestation consistent with a developmental increase in presynaptic vesicles and/or nerve terminals as expected; however, the IR of MAP-2 decreased in all brain regions studied, suggesting concurrent refinement in dendritic branching and spine development. Intermittent UCO as studied with marked but limited hypoxemia resulted in a decrease in IR of SYN for the brain regions of the preterm animals when protein turnover is higher and indicates decreased presynaptic vesicle formation; whereas, MAP-2 IR was selectively increased in the hippocampus CA1 and thalamus of the near-term animals, consistent with reactive dendritic change and heightened vulnerability for neuronal injury. As such, intermittent cord compressions in the ovine fetus can impact protein markers for dendritic and presynaptic nerve development depending on their timing, which might then lead to alterations in synapse formation and neuronal circuitry.

Prevalence of Chlamydia trachomatis and herpes simplex virus type 2: Results from the 2009 to 2011 Canadian Health Measures Survey.

BACKGROUND: Chlamydia, caused by Chlamydia trachomatis, and genital herpes, caused by simplex virus type 2 (HSV-2), are common sexually transmitted infections. Their prevalence has been estimated in selected populations, but overall prevalence in Canada is not known. DATA AND METHODS: Data are from the 2009 to 2011 Canadian Health Measures Survey. Socio-demographic, health and lifestyle information was obtained via a household questionnaire; blood and urine collected at a mobile examination centre were used to identify the presence of Chlamydia trachomatis and HSV-2 among 14- to 59-year-olds. RESULTS: An estimated 13.6% of Canadians (2.9 million) tested positive for HSV-2, and another 0.7% (158,000), for chlamydia. HSV-2 affects higher percentages of women than men, and individuals aged 35 to 59 versus 15 to 34. No significant differences in HSV-2 prevalence were detected by marital status, household income, education, or racial background. Nearly all individuals with laboratory-confirmed chlamydia or HSV-2 were unaware that they were infected. INTERPRETATION: This study is the first in Canada to report laboratory-confirmed prevalence of chlamydia and HSV-2 using a nationally representative sample. Results suggest that most infected people are unaware of their status.

The prevalence of human papillomavirus and its impact on cervical dysplasia in Northern Canada.

INTRODUCTION: Certain types of the Human Papillomavirus (HPV) are sexually transmitted and highly associated with development of cervical dysplasia and cervical cancer but the distribution of HPV infection in the North, particularly amongst First Nations, Metis, and Inuit peoples, is little known. The purposes of the study are to identify the prevalence of type-specific HPV infections and the association of different HPV types with cervical dysplasia among women in Northern Canada. METHODS: This was a cross-sectional study with attendants of the routine or scheduled Pap testing program in the Northwest Territories (NWT), Nunavut, Labrador and Yukon, Canada. Approximately half of each sample was used for Pap test and the remaining was used for HPV genotyping using a Luminex-based method. Pap test results, HPV types, and demographic information were linked for analyses. RESULTS: Results from 14,598 specimens showed that HPV infection was approximately 50% higher among the Aboriginal than the non-Aboriginal population (27.6% vs. 18.5%). Although the most common HPV type detected was HPV 16 across region, the prevalence of other high risk HPV types was different. The age-specific HPV prevalence among Aboriginal showed a 'U' shape which contrasted to non-Aboriginal. The association of HPV infection with cervical dysplasia was similar in both Aboriginal and non-Aboriginal populations. CONCLUSIONS: The HPV prevalence was higher in Northern Canada than in other Areas in Canada. The prevalence showed a higher rate of other high risk HPV infections but no difference of HPV 16/18 infections among Aboriginal in comparison with non-Aboriginal women. This study provides baseline information on HPV prevalence that may assist in surveillance and evaluation systems to track and assess HPV vaccine programs.

Structural proteins during brain development in the preterm and near-term ovine fetus and the effect of intermittent umbilical cord occlusion.

OBJECTIVE: The purpose of this study was to determine the immunoreactivity of selected structural proteins in the preterm and near-term ovine fetal brain and the response to intermittent umbilical cord occlusion as a measure of altered cellular growth. The intermediate filament proteins nestin, vimentin, and glial fibrillary acidic protein was used as markers for astroglial maturation and astrogliosis, and myelin basic protein as a marker for oligodendrocytes and myelin formation. STUDY DESIGN: Fetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days; umbilical cord occlusion was performed in the experimental group by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then killed, and the fetal brain was perfusion fixed and processed for immunohistologic examination of the gray and white matter. Immunoreactivity was quantified with an image analysis system and expressed as the fractional area positive stain for each protein. RESULTS: In both preterm and near-term animal groups, umbilical cord occlusion caused a large decline in arterial Po(2) (to approximately 7 mm Hg), a modest decline in pH (to approximately 7.30), and a modest rise in Pco(2) (to approximately 61 mm Hg; all P <.01), with a return to control values after the occluder release and no cumulative acidosis over each day of study. Vimentin and glial fibrillary acidic protein immunoreactivity showed reciprocal changes, with vimentin decreased and glial fibrillary acidic protein increased in both the gray and white matter of the control group from 0.75 to 0.90 of gestation, which can be attributed to the transition of radial glia into mature astrocytes. Myelin basic protein immunoreactivity increased approximately 3-fold in the white matter of the control group with advancing gestation, which likely reflected active oligodendrocyte differentiation and increased myelination at this time of development. Intermittent umbilical cord occlusion over 4 days resulted in an approximately 60% decrease in nestin, vimentin, and glial fibrillary acidic protein immunoreactivity, which was qualitatively similar for both the gray and white matter and likely indicative of altered protein synthesis and/or degradation, but only in the preterm group and with no change in myelin basic protein immunoreactivity. CONCLUSION: There is considerable change in the immunoreactivity of structural proteins within the ovine fetal brain over the latter part of gestation and consistent with a high rate of protein turnover, as previously reported. Intermittent umbilical cord occlusion as studied with minimal evidence for necrotic cell injury appears capable of altering selected protein synthesis/degradation, more so in younger animals when protein turnover is higher, which might then impact on the brain's development.