An overview of heat stress relief with global warming in perspective.

Global warming seems more probable, whether as gradual warming or increased frequency of warmer episodes. The productivity of cattle in temperate countries will decline unless counteracting steps are adopted. The probability of pre-emptive breeding for maintaining temperate breed performance coupled with heat stress tolerance is too low to be adopted for counteracting warming. The expected warming will mostly involve temperature increases. These will indirectly affect radiant heat gain in animals owing to reduced radiant heat dissipation from the body by convective heat loss, which results in an increased sensitivity to incoming radiant heat at higher air temperatures. These necessitate an emphasis on increasing convective heat loss by structure design and forced air flow by fans. Convective heat loss diminishes with increasing air temperatures. Evaporative heat loss remains the alternative. Evaporative cooling of the ambient requires partial enclosing of the space surrounding the animals and is limited by the humidity in ambient air. An alternative was developed of coupling forced ventilation with wetting of animal surface. The exchange of ambient air flowing on animal surface makes the evaporation practically independent of air humidity and the loss of heat from animal surface practically independent of the surface to air temperature gradient. The coupling of forced ventilation with wetting combination may be attained in various parts of the dairy farm, the holding area of the milking parlour, the feeding trip and the resting area. Each of these requires differing structural and technological adaptations. Climate and farming systems vary between locations which require specific solutions.

Forecasting the general surgery physician workforce deficit in the state of Georgia: a public health perspective.

OBJECTIVES: The aim of the study was to quantify the current shortage of general surgeons in the state of Georgia and to estimate the shortage in 2040. STUDY DESIGN: This is a population-based longitudinal study. METHODS: Data were collected from the US Census Bureau, the Georgia Board for Physician Workforce, and the Accreditation Council for Graduate Medical Education to evaluate changes in the supply of general surgeons from 2000 to 2017 and estimate an expected shortage through 2040. RESULTS: The state of Georgia experienced a net loss of 120 surgeons from 2000 to 2017 and currently faces a shortage of 139 general surgeons. We project a deficit of between 285 and 725 general surgeons in the state of Georgia by 2040. CONCLUSION: Georgia will face a substantial general surgical workforce shortage by 2040. Enhanced efforts at boosting both the output of surgical training programs as well as recruitment and retention of surgeons may help alleviate this deficit.

Implication of Integrin α2ß1 in Proliferation and Invasion of Human Breast Carcinoma and Melanoma Cells: Noncanonical Function of Akt Protein Kinase.

Blocking the expression of integrin α2ß1, which was accomplished by transduction of α2-specific shRNA, resulted in significant inhibition of proliferation and clonal activity in human MCF-7 breast carcinoma and SK-Mel-147 melanoma cells. Along with these changes, deprivation of α2ß1 caused a sharp decrease in melanoma cell invasion in vitro. Analysis of integrin-mediating signal pathways that control cell behavior revealed a significant increase in activity of Akt protein kinase in response to depletion of α2ß1. The increase in Akt activity that accompanies a suppressive effect on cell invasion contradicts well-known Akt function aimed at stimulation of tumor progression. This contradiction could be explained by the "reversed" (noncanonical) role played by Akt in some cells that consists in suppression rather than promotion of invasive phenotype. To test this suggestion, the effects of Akt inhibitors on invasive activity of SK-Mel-147 cells were investigated. If the above suggestion is true, then inhibition of Akt in cells depleted of α2ß1 should result in the restoration of their invasive activity. It appeared that treatment with LY294002, which inhibits all Akt isoforms (Akt1, Akt2, Akt3), not only failed to restore the invasive phenotype of melanoma cells but further attenuated their invasive activity. However, treatment of the cells with an Akt1-specific inhibitor significantly increased their invasion. Thus, the stimulating effect of α2ß1 integrin on invasion of melanoma cells is realized through a mechanism based on inhibition of one of the Akt isoforms, which in these cells exhibits a noncanonical function consisting in suppression of invasion.

Hyperexpression of Integrin α5ß1 Promotes Resistance of MCF-7 Human Breast Carcinoma Cells to Doxorubicin via ERK Protein Kinase Down-regulation.

In MCF-7 human breast carcinoma cells, α5ß1 integrin hyperexpression, which was accomplished by transduction of a full-length α5 integrin cDNA, increased by about 50-70% the number of cells, survived during 48-72 h cell treatment with doxorubicin. Up-regulation of α5ß1 reduced the level of the apoptogenic p53 protein and p21 cell cycle inhibitor, but enhanced the activity of Akt and mTOR protein kinases. In addition to these findings, we observed a significant decrease in the activity of both isoforms of phosphokinase Erk1/2, which is known to play a key role in cell viability pathways, including pathways alleviating stress damages caused by distinct antitumor drugs. Diminished Erk activity accompanying the rise of drug resistance can be explained by an "atypical" function of this kinase, which, in the cells studied, promotes an enhanced rather than reduced sensitivity to doxorubicin. To verify this suggestion, the effect of a specific Erk inhibitor, PD98059, on the resistance to doxorubicin of control and α5 cDNA-transduced MCF-7 cells was investigated. The data showed that suppression of Erk activity increased the resistance of control cells (transduced with an "empty" vector) to a level higher than that demonstrated by the α5 cDNA-transduced cells. The highest level of resistance was observed in α5ß1-trancduced cells treated with PD98059. Akt and mTOR kinase inhibitors had little if any effect on doxorubicin resistance of α5 cDNA-transduced MCF-7 cells. The data show for the first time that integrin α5ß1 can stimulate drug resistance of tumor cells through a mechanism based on the inhibition of protein kinase Erk. From a more general view, the results of this investigation suggest that signal protein kinases can perform in tumor cells "non-canonical" functions, opposite to those, which are the basis for using kinase inhibitors in targeted cancer therapy. It follows that if a protein kinase is supposed to be used as a target for such therapy, it is important to explore its features in the particular tumor prior to the onset of treatment.

A comparison of THI indices leads to a sensible heat-based heat stress index for shaded cattle that aligns temperature and humidity stress.

The combined temperature-humidity heat stress is estimated in farm animals by indices derived of an index based on human thermal comfort sensation. The latter index consists of temperature and humidity measures that sum to form the temperature-humidity index (THI). The hitherto unknown relative contribution of temperature and humidity to the THI was examined. A temperature-humidity data set (temperature 20-42 °C and relative humidity 10-70 %) was used to assess by regression procedures the relative weights of temperature and humidity in the variance of THI values produced by six commonly used heat stress indices. The temperature (Ta) effect was predominant (0.82-0.95 of variance) and humidity accounted for only 0.05 to 0.12 of THI variance, half of the variance encountered in animal responses to variable humidity heat stress. Significant difference in THI values was found between indices in the relative weights of temperature and humidity. As in THI indices, temperature and humidity are expressed in different physical units, their sum has no physical attributes, and empirical evaluations assess THI relation to animal responses. A sensible heat THI was created, in which at higher temperatures humidity reaches 0.25 of sensible heat, similarly to evaporative heat loss span in heat stressed animals. It relates to ambient temperature-humidity similarly to present THI; its values are similar to other THI but greater at higher humidity. In warm conditions, mean animal responses are similar in both indices. The higher sensitivity to humidity makes this index preferable for warm-humid conditions.

MR volumetric assessment of endolymphatic hydrops.

OBJECTIVES: We aimed to volumetrically quantify endolymph and perilymph spaces of the inner ear in order to establish a methodological basis for further investigations into the pathophysiology and therapeutic monitoring of Menière's disease. METHODS: Sixteen patients (eight females, aged 38-71 years) with definite unilateral Menière's disease were included in this study. Magnetic resonance (MR) cisternography with a T2-SPACE sequence was combined with a Real reconstruction inversion recovery (Real-IR) sequence for delineation of inner ear fluid spaces. Machine learning and automated local thresholding segmentation algorithms were applied for three-dimensional (3D) reconstruction and volumetric quantification of endolymphatic hydrops. Test-retest reliability was assessed by the intra-class coefficient; correlation of cochlear endolymph volume ratio with hearing function was assessed by the Pearson correlation coefficient. RESULTS: Endolymph volume ratios could be reliably measured in all patients, with a mean (range) value of 15% (2-25) for the cochlea and 28% (12-40) for the vestibulum. Test-retest reliability was excellent, with an intra-class coefficient of 0.99. Cochlear endolymphatic hydrops was significantly correlated with hearing loss (r = 0.747, p = 0.001). CONCLUSIONS: MR imaging after local contrast application and image processing, including machine learning and automated local thresholding, enable the volumetric quantification of endolymphatic hydrops. This allows for a quantitative assessment of the effect of therapeutic interventions on endolymphatic hydrops. KEY POINTS: • Endolymphatic hydrops is the pathological hallmark of Menière's disease. • Endolymphatic hydrops can be visualized by locally enhanced ultra-high-resolution MR imaging. • Computer-aided image processing enables quantification of endolymphatic hydrops. • Endolymphatic hydrops correlates with hearing loss in patients with Menière's disease. • Therapeutic trials in Menière's disease can be monitored with this quantitative approach.

Implication of α2ß1 integrin in anoikis of MCF-7 human breast carcinoma cells.

Silencing of α2ß1 integrin expression significantly promoted anchorage-dependent apoptosis (anoikis) and drastically reduced clonal activity of MCF-7 human breast carcinoma cells. Depletion of α2ß1 enhanced the production of apoptotic protein p53 and of inhibitor of cyclin-dependent protein kinases, p27, while downregulating antiapoptotic protein Bcl-2 and multifunctional protein cMyc. Blocking the expression of α2ß1 had no effect on activity of protein kinase Akt, but it sharply increased the kinase activity of Erk1/2. Pharmacological inhibition of Erk1/2 had a minor effect on anoikis of control cells, while it reduced anoikis of cells with downregulated α2ß1 to the level of control cells. The data show for the first time that integrin α2ß1 is implicated in the protection of tumor cells from anoikis through a mechanism based on the inhibition of protein kinase Erk.

Inter-animal radiation as potential heat stressor in lying animals.

A model for predicting inter-animal radiant heat exchange in shaded animals is presented, with emphasis on mature cattle. When a cow's surface temperature is 35 °C, as is common in warmer climates, it loses ∼510 Watt m(-2) as radiant heat. Net radiant heat balance depends on radiation coming from bodies in the vicinity. In the 30 °C radiant temperature shaded environment typical of warm climates, net radiant loss from a lactating cow is ∼60 Watt m(-2), i.e., 30 % of its ∼173 Watt m(-2) heat production. Cows rest for 8-14 h day(-1). The heat exchange of a lying cow differs from that of a standing one: the body center is low and 20-30 % of its surface contacts a surface of relatively low heat conductance. Lying reduces the impact of the surrounding shaded area on heat exchange but increases that of heat radiating from neighboring cows. When a cow rests adjacent to other cows, with 1.25 m between body centers when in stalls, it occupies about 140° of the horizontal plane of view. Heat emitted from the animal's surface reduces the net radiant heat loss of a resting cow by ∼30 Watt m(-2). In contrast, the presence of cows at 5 and 10 m distance, e.g., cows resting on straw in loose yard housing, reduces the net radiant heat loss of the resting cow by 9 and 5 Watt m(-2), respectively. Radiant heat input increases with animal density, which is beneficial in cooler climates, but acts as a stressor in warm climates.

Implication of integrin α5ß1 in senescence of SK-Mel-147 human melanoma cells.

Downregulation of α5ß1 integrin in the SK-Mel-147 human melanoma culture model sharply inhibits the phenotypic manifestations of tumor progression: cell proliferation and clonal activity. This was accompanied by a 2-3-fold increase in the content of SA-ß-Gal positive cells thus indicating an increase in the cellular senescence phenotype. These changes were accompanied by a significant increase in the activity of p53 and p21 tumor suppressors and components of the PI3K/Akt/mTOR/p70 signaling pathway. Pharmacological inhibition of mTORC1 reduced the content of SA-ß-Gal positive cells in the population of α5ß1-deficient SK-Mel-147 cells. A similar effect was observed with pharmacological and genetic inhibition of the activity of Akt1, one of the three Akt protein kinase isoenzymes; suppression of other Akt isozymes did not affect melanoma cell senescence. The results presented in this work and previously obtained indicate that α5ß1 shares with other integrins of the ß1 family the function of cell protection from senescence. This function is realized via regulation of the PI3K/Akt1/mTOR signaling pathway, in which Akt1 exhibits a non-canonical activity.

Occult myocardial injury is prevalent amongst elderly patients in the hospital-at-home setting. A retrospective analysis of 213 patients.

Background: Hospital-at-Home (HAH) is a valid alternative for in-hospital stay for a wide variety of clinical indications. Occult myocardial injury, associated with acute illness, mainly occurs in patients with a background of non-obstructive coronary disease. The aim of this study was to describe the prevalence of this phenomenon in our HAH population. Methods: A retrospective description and analysis of data collected for patients admitted to the Sheba beyond's HAH services during 14 months. Results: During a period of 14 months (7/10/21-6/12/22), blood troponin measurements were available for 213 patients (median age 78 years, 52% males) hospitalized mainly for infectious causes. The median HS (highly sensitive) troponin level was 7.7 ng/L (IQR = 13.2 ng/L) (the normal upper limit is 12 ng/L) with 31% of all patients demonstrating an abnormally increased troponin level (68/213). Of all patients, 64% had a background diagnosis of a cardiovascular disease (138/213), of whom, 49% had abnormal HS troponin levels (68/138). No patient suffered from acute cardiac function deterioration and no patient died during their hospital-at-home stay. Conclusion: The prevalence of occult myocardial injury amongst elderly patients admitted to hospital-at-home stay for diagnoses other than myocardial infarction is relatively high but it is not associated with worse short-term clinical outcomes.

High-content microscopy reveals a morphological signature of bortezomib resistance.

Drug resistance is a challenge in anticancer therapy, particularly with targeted therapeutics and cytotoxic compounds. In many cases, cancers can be resistant to the drug prior to exposure, i.e., possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug sensitivity prior to treatment. We therefore isolated clonal cell lines that were either sensitive or resistant to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features typically different between resistant and sensitive clones. These features were compiled to generate a morphological signature of bortezomib resistance, which correctly predicted the bortezomib treatment response in seven of ten cell lines not included in the training dataset. This signature of resistance was specific to bortezomib over other drugs targeting the ubiquitin-proteasome system. Our results provide evidence that intrinsic morphological features of drug resistance exist and establish a framework for their identification.

Lung cancer physicians' referral practices for palliative care consultation.

BACKGROUND: Integration of palliative care with standard oncologic care improves quality of life and survival of lung cancer patients. We surveyed physicians to identify factors influencing their decisions for referral to palliative care. METHODS: We provided a self-administered questionnaire to physicians caring for lung cancer patients at five medical centers. The questionnaire asked about practices and views with respect to palliative care referral. We used multiple regression analysis to identify predictors of low referral rates (<25%). RESULTS: Of 155 physicians who returned survey responses, 75 (48%) reported referring <25% of patients for palliative care consultation. Multivariate analysis, controlling for provider characteristics, found that low referral rates were associated with physicians' concerns that palliative care referral would alarm patients and families [odds ratio (OR) 0.45, 95% confidence interval (CI) 0.21-0.98], while the belief that palliative care specialists have more time to discuss complex issues (OR 3.07, 95% CI 1.56-6.02) was associated with higher rates of referral. CONCLUSIONS: Although palliative care consultation is increasingly available and recommended throughout the trajectory of lung cancer, our data indicate it is underutilized. Understanding factors influencing decisions to refer can be used to improve integration of palliative care as part of lung cancer management.

Radiant heat loss, an unexploited path for heat stress reduction in shaded cattle.

Reducing thermal radiation on shaded animals reduces heat stress independently of other means of stress relief. Radiant heat exchange was estimated as a function of climate, shade structure, and animal density. Body surface portion exposed to radiant sources in shaded environments was determined by geometrical relations to determine angles of view of radiation sources (roof underside, sky, sun-exposed ground, shaded ground) on the animal's surface. The relative representation of environment radiation sources on the body surface was determined. Animal thermal radiation balance was derived from radiant heat gained from radiation sources (including surrounding animals) and that lost from the animal surface. The animal environment was assumed to have different shade dimensions and temperatures. These were summed to the radiant heat balance of the cow. The data formed served to estimate the effect of changes in intensity of radiation sources, roof and shaded surface dimensions, and animal density on radiant heat balance (Rbal) of cattle. Roof height effect was expressed by effect of roof temperature on Rbal. Roof underside temperature (35 to 75°C) effect on Rbal was reduced by roof height. If roof height were 4m, an increase in its underside temperature from 35 to 75°C would increase mean Rbal from -63 to -2 W·m⁻², whereas if roof height were 10 m, Rbal would only increase from -99 to -88 W·m⁻². A hot ground temperature increase from 35 to 65°C reduced mean Rbal heat loss from -45 to 3 W·m⁻². Increasing the surface of the shaded area had only a minor effect on Rbal and on the effect of hot ground on Rbal. Increasing shade roof height reduced the effect of roof temperature on Rbal to minor levels when height was > 8m. Increasing the roof height from 4 to 10 m decreased Rbal from -32 to -94 W·m⁻². Increasing indirect radiation from 100 to 500 W·m⁻² was associated with an increase in Rbal from -135 to +23 W·m⁻². Their combined effects were lower Rbal with increasing roof height and a reduction in rate of decrease with increasing level of indirect radiation. Roof height as an Rbal attenuator declined with increasing indirect radiation level. The latter factor might be reduced by lowering roof surface radiation absorption and through roof heat transfer, as well as by use of shade structure elements to reduce indirect radiation in the shaded area. Radiant heat from the cow body surface may be reduced by lower cow density. Radiant heat attenuation may thus further elevate animal productivity in warm climates, with no associated operation costs.

[Integrin α3ß1 signaling in regulation of the SK-Mel-147 melanoma cell senescence]. / Signaling integrina α3ß1 v regulirovanii stareniia kletok SK-MEL-147 melanomy cheloveka.

Using a model of the human SK-Mel-147 melanoma cell line, it was shown that blocking the expression of integrin α3ß1 by transduction of cells with α3-specific shRNA did not affect their proliferation, but sharply increased the proportion of SA-ß-Gal-positive cells, a phenotypic feature of cell senescence. These findings were accompanied by a significant increase in the activity of the Akt and mTOR protein kinases and the expression of p53 and p21 oncosupressors. Pharmacological inhibition of mTORC1 reduced the number SA-ß-Gal-positive cells in the SK-Mel-147 cell population depleted of α3ß1. Based on our recent data on a non-canonical function of Akt isomers in the regulation of SK-Mel-147 cell senescence caused by deficiency of α2ß1 receptor, we investigated the role of Akt isomers in senescence induced by the α3ß1 knockdown. It appeared that in the cell population with downregulated α3ß1, inhibition of Akt1 reduced the number SA-ß-Gal positive cells to the level of control cell population, while inhibition of Akt2 had no visible effect. Our results demonstrate that the laminin-specific integrin α3ß1, like the collagen-specific receptor α2ß1, is involved in tumor cell protection from senescence, and senescence induced by α3ß1 depletion, like that caused by α2ß1 deficiency, is based on a signaling mechanism employing a non-canonical function of the Akt1 isoform.

Analysis of copy number variation in men with non-obstructive azoospermia.

BACKGROUND: Recent findings demonstrate that single nucleotide variants can cause non-obstructive azoospermia (NOA). In contrast, copy number variants (CNVs) were only analysed in few studies in infertile men. Some have reported a higher prevalence of CNVs in infertile versus fertile men. OBJECTIVES: This study aimed to elucidate if CNVs are associated with NOA. MATERIALS AND METHODS: We performed array-based comparative genomic hybridisation (aCGH) in 37 men with meiotic arrest, 194 men with Sertoli cell-only phenotype, and 21 control men. We filtered our data for deletions affecting genes and prioritised the affected genes according to the literature search. Prevalence of CNVs was compared between all groups. Exome data of 2,030 men were screened to detect further genetic variants in prioritised genes. Modelling was performed for the protein encoded by the novel candidate gene TEKT5 and we stained for TEKT5 in human testicular tissue. RESULTS: We determined the cause of infertility in two individuals with homozygous deletions of SYCE1 and in one individual with a heterozygous deletion of SYCE1 combined with a likely pathogenic missense variant on the second allele. We detected heterozygous deletions affecting MLH3, EIF2B2, SLX4, CLPP and TEKT5, in one subject each. CNVs were not detected more frequently in infertile men compared with controls. DISCUSSION: While SYCE1 and MLH3 encode known meiosis-specific proteins, much less is known about the proteins encoded by the other identified candidate genes, warranting further analyses. We were able to identify the cause of infertility in one out of the 231 infertile men by aCGH and in two men by using exome sequencing data. CONCLUSION: As aCGH and exome sequencing are both expensive methods, combining both in a clinical routine is not an effective strategy. Instead, using CNV calling from exome data has recently become more precise, potentially making aCGH dispensable.

Invited review: Are adaptations present to support dairy cattle productivity in warm climates?

Environmental heat stress, present during warm seasons and warm episodes, severely impairs dairy cattle performance, particularly in warmer climates. It is widely viewed that warm climate breeds (Zebu and Sanga cattle) are adapted to the climate in which they evolved. Such adaptations might be exploited for increasing cattle productivity in warm climates and decrease the effect of warm periods in cooler climates. The literature was reviewed for presence of such adaptations. Evidence is clear for resistance to ticks and tick-transmitted diseases in Zebu and Sanga breeds as well as for a possible development of resistance to ticks in additional breeds. Development of resistance to ticks demands time; hence, it needs to be balanced with potential use of insecticides or vaccination. The presumption of higher sweating rates in Zebu-derived breeds, based upon morphological differences in sweat glands between breeds, has not been substantiated. Relatively few studies have examined hair coat characteristics and their responses to seasonal heat, particularly in temperate climate breeds. Recently, a gene for slick hair coat has been observed that improved heat tolerance when introduced into temperate climate breeds. No solid evidence exists that hair coat in these lines is lighter than in well-fed warm climate-adapted Holsteins. Warm climate breeds and their F1 crosses share as dominant characteristics lower maintenance requirements and milk yields, and limited response to improved feeding and management. These characteristics are not adaptations to a feed-limited environment but are constitutive and useful in serving survival when feed is scarce and seasonal and high temperatures prevail. The negative relationship between milk yield and fertility present in temperate climates breeds also prevails in Zebu cattle. Fertility impairment by warm conditions might be counteracted in advanced farming systems by extra corporeal early embryo culture. In general, adaptations found in warm climate cattle breeds did not increase heat dissipation capacity, but rather diminished climate-induced strain by decreasing milk production. The negative relationship between reproductive efficiency and milk yield, although relatively low, also appears in Zebu cattle. This association, coupled with limited feed intake, acting over millennia, probably created the selection pressure for a low milk production in these breeds.

Biomechanical consequences of compromised elastic fiber integrity and matrix cross-linking on abdominal aortic aneurysmal enlargement.

Abdominal aortic aneurysms (AAAs) are characterized histopathologically by compromised elastic fiber integrity, lost smooth muscle cells or their function, and remodeled collagen. We used a recently introduced mouse model of AAAs that combines enzymatic degradation of elastic fibers and blocking of lysyl oxidase, and thus matrix cross-linking, to study progressive dilatation of the infrarenal abdominal aorta, including development of intraluminal thrombus. We quantified changes in biomaterial properties and biomechanical functionality within the aneurysmal segment as a function of time of enlargement and degree of thrombosis. Towards this end, we combined multi-modality imaging with state-of-the art biomechanical testing and histology to quantify regional heterogeneities for the first time and we used a computational model of arterial growth and remodeling to test multiple hypotheses, suggested by the data, regarding the degree of lost elastin, accumulation of glycosaminoglycans, and rates of collagen turnover. We found that standard histopathological findings can be misleading, while combining advanced experimental and computational methods revealed that glycosaminoglycan accumulation is pathologic, not adaptive, and that heightened collagen deposition is ineffective if not cross-linked. In conclusion, loss of elastic fiber integrity can be a strong initiator of aortic aneurysms, but it is the rate and effectiveness of fibrillar collagen remodeling that dictates enlargement. STATEMENT OF SIGNIFICANCE: Precise mechanisms by which abdominal aortic aneurysms enlarge remain unclear, but a recent elastase plus ß-aminopropionitrile mouse model provides new insight into disease progression. As in the human condition, the aortic degeneration and adverse remodeling are highly heterogeneous in this model. Our multi-modality experiments quantify and contrast the heterogeneities in geometry and biomaterial properties, and our computational modeling shows that standard histopathology can be misleading. Neither accumulating glycosaminoglycans nor frustrated collagen synthesis slow disease progression, thus highlighting the importance of stimulating adaptive collagen remodeling to limit lesion enlargement.

A monoclonal antibody (3G5)-defined ganglioside antigen is expressed on the cell surface of microvascular pericytes.

The identification of microvascular pericytes in vitro relies principally on morphological characteristics and growth dynamics, as there is a paucity of immunochemical markers for these cells. Consequently, an attempt was made to identify mAb reagents that would aid in both the rapid identification and enrichment of retinal capillary pericytes in vascular cell cultures. A panel of mAbs raised by xenogeneic immunization of mice with various tissues was screened for immunoreactivity with dissociated cultures of bovine retinal capillary pericytes. Two antibodies from the panel (3G5 and HISL-8) were seen to react with pericytes by indirect immunofluorescence. The mAb 3G5 was selected for further study. mAb 3G5 did not react with dissociated cultures of smooth muscle cells, endothelial cells, or retinal pigmented endothelial cells. The pericyte 3G5 antigen was insensitive to the action of trypsin; therefore, mAb 3G5 was used to selectively purify pericytes from trypsinized mixed retinal cell cultures by flow cytometry. 3G5+ pericytes (representing 8% of cells in a mixed retinal cell culture) were enriched at least nine-fold to represent greater than 70% of cells. The mAb 3G5 stained retinal capillaries in vivo with a fluorescence distribution consistent with pericyte staining. The 3G5 antigen of cultured pericytes was found to be a glycolipid of mobility intermediate between ganglioside markers GM1 and GM2.