Thermoregulation of the bovine scrotum 2: simulated acute and chronic heat waves reduces the scrotal thermoregulatory capability of Wagyu bulls.

The objective of this study was to investigate the effect of acute and chronic heat load events on scrotal temperature (ST), body temperature (BT) and bull behaviour, and to examine the interrelationship between these parameters; the underlying hypothesis was that adverse heat treatments delivered in a temperature controlled environment will lead to thermoregulatory dysfunction of the bull scrotum. Six sexually mature Wagyu bulls were used in this study with data loggers surgically implanted into the abdominal cavity and scrotum. Body temperate and ST were recorded at 30-min intervals for the duration of the study. There were two housing locations used throughout the study, outdoor pens and climate control rooms. The study was designed as a four-phase crossover design with two heat treatments: (1) a 5-day acute challenge, and (2) a 14-day chronic challenge. The study was also blocked by phase to control for systematic change between phases with a thermoneutral (TN) phase in outdoor pens between each heat challenge. Observations within the climate rooms were conducted at 1-h intervals and data on panting scores (PS), respiration rate (RR), posture (standing or lying) and general behaviours (feeding, drinking, ruminating) recorded. Ambient temperature (AT, °C) and relative humidity (RH, %) were obtained at 10-min intervals and used to calculate the temperature humidity index (THI). Multiple models were conducted using a linear mixed effects model that contained different permutations of date and time factors and interactions as well as inclusion of an autoregressive parameter. The strongest model based on Akaike's information criterion (AIC) was selected and further analysed. Ambient conditions during heat treatments were consistent with heat load and bulls showed typical physiological symptoms of the same. Maximum ST for acute and chronic treatments occurred once AT had exceeded 34 °C for at least 3 h (acute 35.59 °C at 1500 h; chronic 35.18 °C at 1400 h), whereas during TN conditions, maximum ST was at 2100 h. All phases showed variation in ST throughout the day. There were strong cross correlations between ST and RR during the heat treatments (acute r = 0.918, P < 0.0001; chronic r = 0.916, P < 0.0001), but not during TN (r = 0.411, P < 0.05). Our results confirmed that the ST of the bulls used in this study was not held at a constant temperature and that there was a possible connection between ST and RR. We have shown that during a period of heat load, the thermoregulatory mechanisms thought responsible for maintaining bovine ST appear to breakdown.

The impact of weather on the incidence of dark cutting in Australian feedlot cattle.

This study conducted a retrospective analysis of historical Meat Standard Australia (MSA) carcass data in combination with Bureau of Meteorology (BOM) weather data, to evaluate the relationship between climatic conditions prior to feedlot departure on the incidence of dark cutting grain-fed beef. Data records for 2,795,754 carcasses from 17 commercial feedlots over a 6-year period were evaluated within this study. Carcasses were consigned to 16 abattoirs. Weather data from BOM were recorded at 30-min intervals and were obtained from weather stations with the closest proximity to each feedlot. These data were used to calculate the Temperature Humidity Index (THI). Climatic data were amalgamated into daily observations and a series of predictors including ambient temperature (TA, °C), relative humidity (RH, %), wind speed (WS, m/s), rainfall (mm) and THI. In addition, lag interactions from 24 h out to 28 days prior to exiting the feedlot were generated. The incidence of dark cutting was determined as percentage per cohort with an ultimate pH > 5.7. Data were analysed using three models: model 1 included feedlot, abattoir, hormone growth promotant status and sex as fixed effects. Model 2 incorporated the fixed effects within model 1 and minimum, maximum and standard deviation (SD) of TA and RH, daily range in TA, average WS and rainfall as random effects. Model 3 incorporated minimum, maximum, range and SD of THI, average WS and rainfall as random effects in addition to the fixed effects of model 1. The incidence of dark cutting within feedlot had a 10.1% range in estimated means with the lowest incidence was observed at feedlot 17 (0%) and highest incidence at feedlot 10 (10.1%). The inclusion of the climatic variables in model 2 and model 3 accounted for an additional 0.1 to 0.2% of the incidence of dark cutting carcasses. Higher maximum TA, RH and THI in the 3 to 28 days prior to consignment were all associated with an increased incidence of dark cutting (P < 0.05), but not in the 48 h preceding consignment (P > 0.05). Low minimum TA and low THI were also associated with an increase the incidence of dark cutting across all lag periods (P < 0.05). Increased variation in THI and TA in the 48 h prior to consignment increased dark cutting (P < 0.05) while increased standard deviation (SD) of temperature and THI range also increased dark cutting in the 14 and 28 day prior to feedlot exit (P < 0.05). Smaller minimum ranges in TA in the 28 days prior to consignment also reduced dark cutting (P < 0.05). Climatic conditions accounted for a further 0.1 to 0.2% of the incidence of dark cutting, whereas animal management factors, feedlot and abattoir were able to account for 21% of dark cutting. These data suggest that climatic conditions appear to have an inherent role in the incidence of dark cutting, albeit a small impact. Regardless, understanding the influence of climatic conditions on dark cutting allows for the implementation of management strategies within the supply chain to further reduce the impact of climatic conditions on grain-fed cattle.

The influence of shade availability on the effectiveness of the Dairy Heat Load Index (DHLI) to predict lactating cow behavior, physiology, and production traits.

Numerous climatic indices have been utilized to predict the effect of hot, and cold, climatic conditions on animal production and welfare. To date, the dairy industry has relied extensively on the Temperature Humidity Index (THI) to predict adverse climatic conditions; however, neither solar radiation nor air movement is accounted for in the THI equation. The Dairy Heat Load Index (DHLI) was initially developed as an alternative climate index. In its current format, the DHLI does not account for the effects of heat load mitigation strategies, such as shade, which decreases the negative effects of hot climatic conditions on lactating cows. Therefore, this experiment aimed to determine the effectiveness of the DHLI as a predictor of heat load responses in both shaded and unshaded cows, as compared with the THI. Forty lactating Holstein Friesian (n = 40) cows were selected and paired based on live weight, milk yield, and days in milk. One cow from each pair was randomly allocated to one of two treatments: shaded (n = 20) or unshaded (n = 20). Cows were given 7 days to acclimate prior to the commencement of data collection. After 28 days, cows were transitioned into the alternate treatment in a crossover design and given 7 days to acclimate prior to data collection. Behavioral observations (0800, 1200, 1400, and 1800 h daily), daily milk yield (kg), milk composition (various days), and vaginal temperature (TVAG, °C; 5 pairs/week, over a 4-week rotation) were recorded. Overall, data from this experiment indicated that the DHLI was a better predictor of standing and feeding behaviors in unshaded cows and drinking behaviors in shaded cows. Conversely, the THI was a better predictor of standing behavior and shade usage in shaded cows. Furthermore, the THI was a better predictor of mean panting score (MPS) in shaded cows, whereas the DHLI performed better in unshaded cows. Additionally the DHLI was a better predictor of TVAG in these cows. Finally, when evaluating the 7-day average of each climatic index, the DHLI was a better predictor of change in milk yield. Incorporation of additional animal and management factors is required if the DHLI is to become an effective heat load management tool.

Influence of feeding Saccharomyces cerevisiae on the heat load responses of lactating dairy cows during summer.

The objective of this study was to evaluate the influence of supplementing lactating dairy cows with Saccharomyces cerevisiae on milk production and composition, cow behavior, and physiological responses during summer. Twenty primiparous cows were used and two treatments were imposed: (1) control (CON); and (2) probiotic supplementation (PRO; S. cerevisiae, providing 1010 colony forming units (CFU) per day). Rumen temperature (TRUM, °C) and pH were obtained via rumen boluses. Rumen temperatures were obtained from all cows (n = 20) at 10-min intervals and ruminal pH were obtained from five cow pairs (n = 10) at 10-min intervals. Ambient temperature (TA; °C), relative humidity (RH; %), wind speed (WS; m/s), and solar radiation (SR; W/m2) were recorded at 10-min intervals. The temperature humidity index (THI) was calculated using TA and RH. Cows were milked twice daily. Milk fat (%), protein (%), lactose (%), and somatic cell count (SCC, '000) were evaluated on 16 occasions. Cows were observed three times (0800 h; 1200 h; and 1400 h) daily for panting score (PS); respiration rate (RR); posture (standing/lying); shade utilization; and cow activity (eating/drinking/ruminating). Individual PS were used to calculate a mean panting score (MPS) for CON and PRO treatments for each observation. S. cerevisiae did not influence milk yield (P = 0.87), fat (P = 0.82), protein (P = 0.26) or SCC (P = 0.19), although there was a tendency for PRO cows to have higher lactose (P = 0.06). Probiotics did not influence the proportion of cows utilizing shade (P = 0.42); standing (P = 0.41); ruminating (P = 0.72); or drinking (P = 0.40). All cows exhibited an increase in RR (> 24 bpm) at 1200 h and RR showed a steady increase as THI increased (P < 0.0001), regardless of treatment (P = 0.96). Both CON (35.8%) and PRO (40.2%) exhibited an increase in MPS as THI increased from thermoneutral (THI ≤ 74) to very hot (THI ≥ 84.1; P < 0.001). However, PRO cows had lower (2.19 ± 0.09; P < 0.0001) MPS compared with CON (2.54 ± 0.22) cows when THI was categorized as very hot (THI ≥ 84.1). Rumen pH were not influenced by treatment (P = 0.38), however TRUM of PRO cows were 0.2 °C lower across days (P < 0.0001) and hours (P < 0.0001). These results suggest that supplementing cows with S. cerevisiae may support thermoregulation via decreased TRUM and MPS; however, further studies are required.

Heat stress and poultry production: impact and amelioration.

Globally, the poultry industry is gaining significant importance among the agricultural and its allied sectors. However, heat stress was found to negatively affect the poultry production particularly in the tropical regions. This review is therefore an attempt to generate information pertaining to the impacts of heat stress on poultry production and its amelioration. Heat stress reduces the growth, reproductive performance, and egg production in poultry birds. The reduction in productive potential of poultry birds on exposure to heat stress may be attributed to the deviation of energy resources from production to adaptation pathway. There are different approaches pertaining to relieving the adverse impacts of heat stress on poultry production. These approaches can be broadly categorized under genetic, management, and nutritional strategies. These approaches may reduce the negative effects of heat stress and enhance the productive performance of poultry birds. The management strategies include appropriate shelter design, providing shade, using sprinklers, implementing cooling devices, and using fans and ventilation systems. The recommended floor space for mature birds weighing 1.7 kg is 0.06 m2/bird while it is 0.13 m2/bird for the birds weighing 3.5 kg with 27.8 kg/m2 bird density in either case. The nutritional interventions comprise ration balancing and providing essential micronutrients to improve the productive and reproductive performance in poultry birds. Fat, antioxidants, yeast, and electrolyte supplementations are some of the most commonly used nutritional strategies to ensure optimum production in the poultry industry. Furthermore, providing adequate water supply and disease surveillance measures may help to ensure optimum meat and egg production in the birds. The advanced biotechnological tools may aid to identify suitable genetic markers in poultry birds which might help in developing new strains of higher thermo-tolerance by designing suitable breeding program involving marker-assisted selection. These strategies may help to optimize and sustain poultry production in the changing climate scenario.

Development of a reference material for analysing naturally occurring radioactive material from the steel industry.

Accurate measurement of naturally occurring radionuclides in blast furnace slag, a by-product of the steel industry, is required for compliance with building regulations where it is often used as an ingredient in cement. A matrix reference blast furnace slag material has been developed to support traceability in these measurements. Raw material provided by a commercial producer underwent stability and homogeneity testing, as well as characterisation of matrix constituents, to provide a final candidate reference material. The radionuclide content was then determined during a comparison exercise that included 23 laboratories from 14 countries. Participants determined the activity per unit mass for 226Ra, 232Th and 40K using a range of techniques. The consensus values obtained from the power-moderated mean of the reported participant results were used as indicative activity per unit mass values for the three radionuclides: A0(226Ra) = 106.3 (34) Bq·kg-1, A0(232Th) = 130.0 (48) Bq·kg-1 and A0(40K) = 161 (11) Bq·kg-1 (where the number in parentheses is the numerical value of the combined standard uncertainty referred to the corresponding last digits of the quoted result). This exercise helps to address the current shortage of NORM industry reference materials, putting in place infrastructure for production of further reference materials.

Heat load increases the risk of clinical mastitis in dairy cattle.

The study was aimed at assessing heat load-related risk of clinical mastitis (CM) in dairy cows. Records of CM for the years 2014 and 2015 were obtained from a large conventional dairy farm milking about 1,200 Holstein cows in central Italy. A case of CM was defined by the presence of clinical signs and veterinary confirmation. Quarter milk samples were collected and bacteriological investigated for each CM. Etiological agents were identified and classified as environmental or contagious pathogens. Hourly weather data from the nearest weather station were used to calculate heat load index (HLI). Upper and lower thresholds of HLI, at which the animal accumulates or dissipates heat, were settled and used to measure heat load balance through the accumulated heat load (AHL) model. Zero and positive values of AHL indicate periods of thermo-neutral and heat accumulation, respectively. Each case of CM was associated with HLI-AHL values recorded 5 d before the event. The risk of CM was evaluated using a case-crossover design. A conditional logistic regression model was used to calculate the odds ratio and 95% confidence intervals of CM recorded in thermo-neutral (AHL = 0) or heat load (AHL > 0) days, pooled or stratified for pathogen type (environmental or contagious). Classes of AHL as low (<6.5), medium (6.6-34.9), and high (>35) were included in the model. Other variables included in the model were milk yield as liters (<20, 20-30, and >30), days in milk (<60, 60-150, and >150), and parity (1, 2-3, and >3). A total of 1,086 CM cases were identified from 677 cows. Escherichia coli, Streptococcus spp., and Streptococcus uberis were the environmental pathogens isolated with the highest frequency; Staphylococcus aureus prevailed within contagious species. The analysis of pooled data indicated a significant effect of heat load on the occurrence of CM in the contagious pathogen stratum. Higher milk yield, middle and late stage of lactation, and older parity increased the risk of CM under heat load conditions. However, the association between pathogen type and these factors was not clear because the model provided significant odds ratios within all pathogen categories. The present study provided the first evidence of an association between HLI and CM in dairy cattle and suggested the ability of the AHL model to assess the risk of mastitis associated with heat load.

The impact of heat stress on the immune system in dairy cattle: A review.

Heat stress is well documented to have a negative influence on livestock productivity and these impacts may be exacerbated by climate change. Dairy cattle can be more vulnerable to the negative effects of heat stress as these adverse impacts may be more profound during pregnancy and lactation. New emerging diseases are usually linked to a positive relationship with climate change and the survival of microrganisms and/or their vectors. These diseases may exaggerate the immune suppression associated with the immune suppressive effect of heat stress that is mediated by the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-adrenal-medullary (SAM) axes. It has been established that heat stress has a negative impact on the immune system via cell mediated and humoral immune responses. Heat stress activates the HPA axis and increases peripheral levels of glucocorticoids subsequently suppressing the synthesis and release of cytokines. Heat stress has been reported to induce increased blood cortisol concentrations which have been shown to inhibit the production of cytokines such as interleukin-4 (IL-4), IL-5, IL-6, IL-12, interferon γ (IFNγ), and tumor necrosis factor-α (TNF- α). The impact of heat stress on the immune responses of dairy cows could be mediated by developing appropriate amelioration strategies through nutritional interventions and cooling management. In addition, improving current animal selection methods and the development of climate resilient breeds may support the sustainability of livestock production systems into the future.

Prediction models, assessment methodologies and biotechnological tools to quantify heat stress response in ruminant livestock.

Livestock industries have an important role in ensuring global food security. This review discusses the importance of quantifying the heat stress response of ruminants, with an emphasis on identifying thermo-tolerant breeds. There are numerous heat stress prediction models that have attempted to quantify the response of ruminant livestock to hot climatic conditions. This review highlights the importance of investigating prediction models beyond the temperature-humidity index (THI). Furthermore, this review highlights the importance of incorporating other climatic variables when developing prediction indices to ensure the accurate prediction of heat stress in ruminants. Prediction models, particularly the heat load index (HLI) were developed to overcome the limitations of the THI by incorporating ambient temperature (AT), relative humidity (RH), solar radiation (SR) and wind speed (WS). Furthermore refinements to existing prediction models have been undertaken to account for the interactions between climatic variables and physiological traits of livestock. Specifically, studies have investigated the relationships between coat characteristics, respiration rate (RR), body temperature (BT), sweating rate, vasodilation, body weight (BW), body condition score (BCS), fatness and feed intake with climatic conditions. While advancements in prediction models have been occurring, there has also been substantial advancement in the methodologies used to quantify animal responses to heat stress. The most recent development in this field is the application of radio frequency identification (RFID) technology to record animal behaviour and various physiological responses. Rumen temperature measurements using rumen boluses and skin temperature recording using infrared thermography (IRT) are making inroads to redefine the quantification of the heat stress response of ruminants. Further, this review describes several advanced biotechnological tools that can be used to identify climate resilient breeds of ruminant livestock.

A panting score index for sheep.

When exposed to hot conditions, heat dissipation via an increase in respiration rate (RR) is an important thermoregulatory mechanism for sheep. However, evaluating RR under field conditions is difficult. In cattle, a viable alternative has been to assess panting score (PS); therefore, the objective of this study was to evaluate the relationship between RR and PS to determine if a PS index can be used to evaluate heat load in sheep. One hundred and forty-four Merino wethers (44.02 ± 0.32 kg) were used within a climate-controlled study. The study was replicated twice over 29 days, where each replicate consisted of two treatments: (1) thermoneutral (TN) and (2) hot (HOT). Ambient temperature (TA) and relative humidity (RH) were maintained between 18 and 20 °C and 60 and 70% respectively for the TN treatment. For the HOT treatment, heat load increased steadily over the 29 days. Minimum TA was 22.5 °C and maximum was 38.5 °C, while RH decreased (60 to 30%) as TA increased in the HOT treatment. A comprehensive PS classification was developed by enhancing the current sheep PS index and aligning the descriptors with the current PS index utilized in beef cattle studies. Respiration rate and PS were obtained for each animal at 3-h intervals between 0800 h and 1700 h daily. These data were used to determine the mean RR for each PS, across the study and within the TN and HOT treatments. The relationship between PS and RR was evaluated using a Pearson's correlation coefficient. Data were also analyzed using a general linear model to determine the impact of PS, posture and animal identification (animal ID) on RR within each PS. Unsurprisingly, RR increased as PS increased, and PS, 0 and RR, 2.5 were 30.7 ± 0.59 and 246.8 ± 12.20 bpm respectively. There was a strong relationship between RR and PS (r = 0.71; P < 0.0001). As RR increased, sheep were more likely to be observed standing (P < 0.001). Mean PS of sheep within the HOT treatment (1.49 ± 0.02) were greater (P = 0.0085) when compared to the TN (1.17 ± 0.02) sheep. Individual animal ID accounted for approximately 7-37% of the variation observed for RR across PS, indicating that animal ID and climatic conditions were influencing RR and PS. These results suggest that the comprehensive PS index described here can be used as a visual appraisal of the heat load status of sheep.

Effect of heat stress on rumen temperature of three breeds of cattle.

Thirty-six steers (12 of each Angus, Charolais, and Brahman) with an initial BW of 318.5 ± 6.7 kg were used in a 130-day study. Two treatments were imposed: un-shaded and shaded (3 m2/animal; 90% solar block shade cloth). On day 1, steers were administered with rumen temperature boluses. Rumen temperatures (T RUM) were obtained at 10 min intervals over the duration of the study to determine differences in T RUM between Bos indicus and Bos taurus cattle. Six feedlot pens (162 m2) were used with six steers (2/breed) per pen with three pens/treatment. Ambient dry bulb temperature (T A; °C), relative humidity (RH; %), wind speed (WS; m/s) and direction, and solar radiation (SR; W/m2) were recorded at 10 min intervals. Rainfall (mm) was collected daily at 0900 h. From these data, black globe temperature (BGT; °C), temperature humidity index (THI), heat load index (HLI), and accumulated heat load (AHL) were calculated. Individual T RUM were converted to an hourly average and then mean hourly T RUM were converted to a mean within hour T RUM across the 130 days. Rumen temperatures were analyzed using an autoregressive repeated measures model. The model analyzed the effect of breed (P < 0.0002), treatment (P = 0.3543), time of day (hour, h; P < 0.0001), breed × treatment (P < 0.3683), breed × h (P < 0.0001), treatment × h (P < 0.0001), breed × treatment × h (P = 0.0029), pen within treatment (P = 0.0195), and animal × breed × treatment within pen (P = 0.1041). Furthermore, there were breed × treatment × hour differences in T RUM (P = 0.0036), indicating that Bos indicus and Bos taurus regulate T RUM differently.

Thermoregulation of the bovine scrotum 1: measurements of free-range animals in a paddock and pen.

The bull's scrotum and scrotal cord vasculature has traditionally been regarded as a thermoregulatory device for maintaining optimal testicular temperature for normal spermatogenesis. This assumption has mostly been derived from discrete measurements using thermocouples with limited data correlating continuous scrotal temperature (ST) to body temperature (BT). From mid-summer to early autumn, four Wagyu bulls (9-18 months) were surgically implanted with two data loggers (DL) logging at 30 min intervals: one on the right hand side flank and the other was attached to the visceral vaginal tunic of the mid-testis. Bulls were firstly housed in a paddock (PK) for 13 days and then moved to individual pens (IP), again for 13 days. Repeated measures analysis modelled the long-term and diurnal trends in BT and ST. While both day and time of day (TOD) were significant effects for ST at both housing locations (P < 0.005), only TOD showed significance for BT at both locations (P < 0.0001). Significant effects were seen between bulls with ST (F = 167.2, P < 0.001) but not BT (F = 0.03, P = 0.863), suggestive of variation in individual bull thermoregulatory capacity. Dual peaks were observed in ST at 0500 and 2130 h when housed in PK but not IP, suggesting ST may be influenced by external stimuli such as postural or behavioural changes. Reporting concurrent and continuous BT and ST will allow further investigation into factors influencing bovine ST and should be useful in selecting bulls with high degrees of thermoregulation capacity.

Isolation of Nocardia mexicana from focal proliferative tenosynovitis and arthritis in a steer.

CASE REPORT: An 18-month-old Charolais steer was presented with lameness and fluctuant swelling of the right stifle joint, which yielded neutrophils on fine-needle aspiration. A diagnosis of bacterial proliferative tenosynovitis and arthritis was made on postmortem and histological examination. Culture and 16S rRNA sequencing identified a Nocardia sp. with 99% homology with the corresponding DNA fragment of N. mexicana DSM 44952. Antimicrobial susceptibility testing revealed the isolate was susceptible to co-trimoxazole and third-generation cephalosporins. CONCLUSION: We report the first case, both in Australia and internationally, of proliferative tenosynovitis and arthritis caused by Nocardia spp. infection in a bovine and the first report of pathology attributed to N. mexicana in a veterinary patient. Given the limited susceptibility of the bacteria, the poor antimicrobial penetration that would be expected and the morphological changes that had taken place in the joint; the steer would have required protracted antimicrobial treatment in addition to invasive debridement of the lesion. This case emphasises the importance of routinely performing cytology and extended incubation of cultures in cases of arthritis in order to make ethical and economically viable treatment decisions.

Reversible and irreversible non-internalized LDL and methyl LDL accumulation by human fibroblasts.

In previous in vivo animal studies, we showed that low density lipoprotein (LDL) accumulated irreversibly at the edges of healing arterial lesions rather than being internalized and degraded. To see if similar LDL accumulation occurs in vitro, fibroblasts from normal and homozygous familial hypercholesterolemic (FH) subjects were incubated at 37 degrees C with 125I-LDL and 125I-methyl LDL; the latter is not recognized by any known LDL receptor. Normal fibroblast accumulation of LDL and methyl LDL (5 microg/ml) plateaued within 1 h at 200 and 100 ng/mg, respectively. With FH cells, both LDL and methyl LDL accumulation plateaued at 100 ng/mg. Lipoprotein accumulation by both cell types rose steeply at concentrations up to 15-25 microg/ml, and less so at higher concentrations. Except for degradation of LDL by normal cells, degradation was minimal, which indicated that much of the lipoprotein accumulation was unaccompanied by internalization. The accumulation of both lipoproteins by both cell types was greater at 37 degrees C than at 4 degrees C, and was inhibited between 43 and 75% by homologous unlabeled lipoprotein. To see if any accumulation was irreversible, cells were incubated with radiolabeled lipoproteins for 3 h (pulse), then with homologous unlabeled lipoproteins for up to 20 h (chase). About 50% of intact radiolabeled lipoprotein rapidly dissociated from cells into the medium in the first 4 h of the chase period. In contrast, between 4 and 20 h, most of the remaining intact LDL and methyl LDL appeared to be irreversibly bound, because it was released at a rate of only 0-1%/h. Thus, we conclude that, under the conditions studied, both reversible and irreversible non-internalized LDL binding play a major role in LDL accumulation by cultured cells.

Non-pharmacological lowering of low-density lipoprotein by apheresis and surgical techniques.

In the past year, new data have appeared on the long-term benefits of low-density lipoprotein apheresis in severely hypercholesterolemic patients who are refractory to lipid-lowering drug therapy. Such data are critical for clinical decision-making, because they confirm the hypothesis that the dramatic reduction in low-density lipoprotein made possible by this technique produces clear-cut clinical benefits. Because of its efficacy and low incidence of side-effects, apheresis for severe drug-refractory hypercholesterolemia has superseded surgical approaches, such as liver transplantation or ileal bypass.

Biodistribution and autoradiographic localization of I-125--labeled synthetic Peptide in aortic atherosclerosis in cholesterol-fed rabbits.

I-125 labeled SP4 is a synthetic oligopeptide derived from apolipoprotein B of low-density lipoprotein that has been shown to localized in atherosclerotic plaques in experimental animals. However, its biodistribution and mechanism of localization need to be further elucidated. Twenty-four cholesterol-fed (CF) and 20 normal (NL) New Zealand White rabbits were injected with I-125-SP4 and killed 15 to 30 min (6 NL; 6 CF) or 2 h (14 NL; 18 CF) later. We obtained aortic autoradiograms and activity concentrations (% injected dose/gm) in aortic segments and other tissues. The uptake of I-125-SP4 was higher in CF than in NL rabbits in all aortic segments (p < 0.05). I-125-SP4 was cleared rapidly in both CF and NL rabbits with 60 to 70% of the injected dose cleared from the blood by 1 h. No statistically significant differences in radiotracer biodistribution were observed between NL and CF rabbits although activity tended to be higher in the liver, gallbladder, and intestine in NL rabbits and in the kidney and spleen in CF rabbits. Silver grains were distributed mainly on foam cells of the fatty streaks in aortic microautoradiograms from two additional rabbits that had been injected with I-125-SP4. There were 23,518 plus minus 15,878 (SD) grains/mm(2) in fatty plaques but only 14,669 plus minus 11,035 grains/mm(2) in media muscle (p < 0.0001 [9 sections, 17 areas evaluated] in an atherosclerotic animal) in injected animals and 13,439 plus minus 5,565 grains/mm(2) in media muscle (two sections, four areas) in the normal control animals (NS versus media of atherosclerotic animal). I-125-SP4 specifically localizes in aortic atherosclerotic plaques in CF rabbits. There is no significant difference in tissue distribution between normal and CF rabbits except in the aorta. Preliminarily, it appears that the site of tracer uptake is on foam cells and this suggests the possibility of relative specificity for fatty plaque.

Treatment of hypercholesterolemia with heparin-induced extracorporeal low-density lipoprotein precipitation (HELP).

Familial hypercholesterolemia (FH) can cause early disability and death from premature atherosclerotic cardiovascular disease. Patients homozygous for the disease have very high plasma cholesterol, extensive xanthomatosis, and die from atherosclerosis in childhood or early adulthood. Past attempts to improve the prognosis included removal of cholesterol from the circulation by ileal bypass or biliary diversion. Neither treatment was successful. Direct removal by plasmapheresis of low-density lipoprotein (LDL), the primary carrier of cholesterol in plasma, was first performed on an FH homozygous patient in 1966. The treatment was well tolerated and led to rapid diminution of xanthomas. Other experimental treatments included selective LDL apheresis with monoclonal or polyclonal antibody affinity columns. A method for selective LDL apheresis was developed in 1983 by Armstrong, Seidel, and colleagues based on heparin precipitation of LDL at low pH. This method, called HELP, removes all apolipoprotein B-containing lipoproteins including LDL and lipoprotein (a), as well as some fibrinogen. LDL apheresis by HELP is well tolerated; the incidence of side effects is low, and the treatment has been associated with regression of cardiovascular disease. LDL apheresis, rather than liver transplantation, is the treatment of choice for patients with severe, life-threatening hypercholesterolemia which does not respond to diet and drug therapy.

External imaging of atherosclerosis in rabbits using an 123I-labeled synthetic peptide fragment.

The oligopeptide fragment of apolipoprotein B, SP-4, has demonstrated pronounced uptake in the healing edges of balloon-injured rabbit aortic endothelium. To assess 123I-labeled SP-4 for identification of atherosclerotic plaques by gamma camera imaging, 14 Watanabe heritable hyperlipidemic (WHHL) and 5 normal rabbits were imaged 5 minutes and 12 and 24 hours after intravenous injection of 123I-SP-4. In addition, two WHHL and two normal rabbits were injected with 125I-SP-4 for autoradiography. Twelve of the 14 WHHL, but none of the normal, rabbits had visually apparent focal radioiodine accumulation in the region of the aorta. Focus-to-lung and focus-to-heart count ratios were 2.4 +/- 1.3 and 1.0 +/- 0.4, respectively. Five of the visually positive WHHL rabbits were reimaged 4 and 8 weeks later with 123I-NaI and 123I-SP-2 (an apo E peptide), respectively, as negative controls. Perceptible, but faint, aortic localization of 123I-NaI and of 123I-SP-2 was seen in only one animal each. The distributions of atherosclerotic lesions on photographs of the opened WHHL aortas and of film blackening on 125I-SP-4 autoradiograms were identical. In contrast, the two normal rabbit aortas did not exhibit plaques on photographs or film blackening on autoradiograms. Thus, in an animal model closely simulating human atherosclerotic disease, SP-4 localizes specifically in aortic atherosclerotic lesions.

Low-density lipoprotein modification and arterial wall accumulation in a rabbit model of atherosclerosis.

Chemically or enzymatically modified low-density lipoproteins (LDL), with and without changes in surface charge, were studied in vivo in the healing, balloon catheter-deendothelialized rabbit aorta to determine the effect of LDL modification on its accumulation in arterial lesions. In this model, in which healing (reendothelialization) proceeds radially outward from individual aortic branch arteries, it was previously shown by autoradiography that two kinetically distinct compartments accumulated 125I-labeled LDL. In aortic regions which were still deendothelialized, accumulation was diffuse and labile. In contrast, at the edges of the islands of regenerating endothelium, LDL accumulation was intensely focal, as it is in human atherosclerotic lesions, and persisted for at least 40 h after injection in spite of falling levels of radiolabeled LDL in plasma [Chang, M. Y., et al. (1992) Arterioscler. Thromb. 12, 1088-1098]. In the present study, modified LDLs with gradations in charge change were prepared to clarify the role of changes in surface charge on focal aortic LDL accumulation. Oxidized LDL (weakly anionized), desialated LDL (weakly cationized), and reductively methylated LDL (no change in net charge) all accumulated focally. Focal accumulation of native LDL also occurred in ballooned rabbits fed probucol to inhibit LDL oxidation. Strongly anionized succinylated and diazobenzenearsonylated LDL and strongly cationized dimethylpropanediamine LDL did not accumulate focally. The results support the concept that focal sequestration of LDL in arterial lesions is mediated by specific, oxidation-independent patterns of charge and polarity on LDL which are disrupted by major changes in LDL surface charge.