Ultrastructural characterization of host-parasite interactions of Plasmodium coatneyi in rhesus macaques.

Plasmodium coatneyi has been proposed as an animal model for human Plasmodium falciparum malaria as it appears to replicate many aspects of pathogenesis and clinical symptomology. As part of the ongoing evaluation of the rhesus macaque model of severe malaria, a detailed ultrastructural analysis of the interaction between the parasite and both the host erythrocytes and the microvasculature was undertaken. Tissue (brain, heart and kidney) from splenectomized rhesus macaques and blood from spleen-intact animals infected with P. coatneyi were examined by electron microscopy. In all three tissues, similar interactions (sequestration) between infected red blood cells (iRBC) and blood vessels were observed with evidence of rosette and auto-agglutinate formation. The iRBCs possessed caveolae similar to P. vivax and knob-like structures similar to P. falciparum. However, the knobs often appeared incompletely formed in the splenectomized animals in contrast to the intact knobs exhibited by spleen intact animals. Plasmodium coatneyi infection in the monkey replicates many of the ultrastructural features particularly associated with P. falciparum in humans and as such supports its use as a suitable animal model. However, the possible effect on host­parasite interactions and the pathogenesis of disease due to the use of splenectomized animals needs to be taken into consideration.

Towards elimination of HIV transmission, AIDS and HIV-related deaths in the UK.

OBJECTIVES: Our objective was to present recent trends in the UK HIV epidemic (2007-2016) and the public health response. METHODS: HIV diagnoses and clinical markers were extracted from the HIV and AIDS Reporting System; HIV testing data in sexual health services (SHS) were taken from GUMCAD STI Surveillance System. HIV data were modelled to estimate the incidence in men who have sex with men (MSM) and post-migration HIV acquisition in heterosexuals. Office for National Statistics (ONS) data enabled mortality rates to be calculated. RESULTS: New HIV diagnoses have declined in heterosexuals as a result of decreasing numbers of migrants from high HIV prevalence countries entering the UK. Among MSM, the number of HIV diagnoses fell from 3570 in 2015 to 2810 in 2016 (and from 1554 to 1096 in London). Preceding the decline in HIV diagnoses, modelled estimates indicate that transmission began to fall in 2012, from 2800 [credible interval (CrI) 2300-3200] to 1700 (CrI 900-2700) in 2016. The crude mortality rate among people promptly diagnosed with HIV infection was comparable to that in the general population (1.22 vs. 1.39 per 1000 aged 15-59 years, respectively). The number of MSM tested for HIV at SHS increased annually; 28% of MSM who were tested in 2016 had been tested in the preceding year. In 2016, 76% of people started antiretroviral therapy within 90 days of diagnosis (33% in 2007). CONCLUSIONS: The dual successes of the HIV transmission decline in MSM and reduced mortality are attributable to frequent HIV testing and prompt treatment (combination prevention). Progress towards the elimination of HIV transmission, AIDS and HIV-related deaths could be achieved if combination prevention, including pre-exposure prophylaxis, is replicated for all populations.

Monitoring the HIV continuum of care in key populations across Europe and Central Asia.

OBJECTIVES: The aim of the study was to measure and compare national continuum of HIV care estimates in Europe and Central Asia in three key subpopulations: men who have sex with men (MSM), people who inject drugs (PWID) and migrants. METHODS: Responses to a 2016 European Centre for Disease Prevention and Control (ECDC) survey of 55 European and Central Asian countries were used to describe continuums of HIV care for the subpopulations. Data were analysed using three frameworks: Joint United Nations Programme on HIV/AIDS (UNAIDS) 90-90-90 targets; breakpoint analysis identifying reductions between adjacent continuum stages; quadrant analysis categorizing countries using 90% cut-offs for continuum stages. RESULTS: Overall, 29 of 48 countries reported national data for all HIV continuum stages (numbers living with HIV, diagnosed, receiving treatment and virally suppressed). Six countries reported all stages for MSM, seven for PWID and two for migrants. Thirty-one countries did not report data for MSM (34 for PWID and 41 for migrants). In countries that provided key-population data, overall, 63%, 40% and 41% of MSM, PWID and migrants living with HIV were virally suppressed, respectively (compared with 68%, 65% and 68% nationally, for countries reporting key-population data). Variation was observed between countries, with higher outcomes in subpopulations in Western Europe compared with Eastern Europe and Central Asia. CONCLUSIONS: Few reporting countries can produce the continuum of HIV care for the three key populations. Where data are available, differences exist in outcomes between the general and key populations. While MSM broadly mirror national outcomes (in the West), PWID and migrants experience poorer treatment and viral suppression. Countries must develop continuum measures for key populations to identify and address inequalities.

Significant differences in maternal child-feeding style between ethnic groups in the UK: the role of deprivation and parenting styles.

BACKGROUND: Nonresponsive maternal child-feeding interactions, such as restricting, pressurising and emotional feeding, can affect the ability of a child to self-regulate intake and increase the risk of becoming overweight. However, despite findings that South Asian and Black children living in the UK are more likely to be overweight, UK research has not considered how maternal child-feeding style might differ between ethnic groups. The present study aimed to explore variations in maternal child-feeding style between ethnic groups in the UK, taking into account associated factors such as deprivation and parenting style. METHODS: Six hundred and fifty-nine UK mothers with a child who was aged 5-11 years old completed a questionnaire. Items included ethnicity and demographic data, as well as copies of the Child Feeding Questionnaire, Parental Feeding Styles Questionnaire and Parenting Styles and Dimensions Questionnaire. RESULTS: Significant differences in perceived responsibility (P = 0.002), restriction (P = 0.026), pressure to eat (P = 0.045), instrumental feeding (P = 0.000) and emotional feeding (P = 0.000) were found between the groups. Mothers from South Asian backgrounds reported higher levels of pressure to eat, emotional feeding and indulgent feeding styles, whereas mothers from Chinese backgrounds reported greater perceived responsibility and restriction. Mothers from Black and White British backgrounds were not significantly higher with respect to any behaviour. Maternal child-feeding style was also associated with deprivation and parenting style, although these did not fully explain the data. CONCLUSIONS: Understanding cultural factors behind maternal child-feeding style, particularly around pressurising and indulgent feeding behaviours, may play an important part in reducing levels of children who are overweight and obese in the UK.

People with diagnosed HIV infection not attending for specialist clinical care: UK national review.

BACKGROUND: Regular clinical care is important for the well-being of people with HIV. We sought to audit and describe the characteristics of adults with diagnosed HIV infection not reported to be attending for clinical care in the UK. METHODS: Public Health England (PHE) provided clinics with lists of patients diagnosed or seen for specialist HIV care in 2010 but not linked to a clinic report or known to have died in 2011. Clinics reviewed case-notes of these individuals and completed questionnaires. A nested case-control analysis was conducted to compare those who had remained in the UK in 2011 while not attending care with individuals who received specialist HIV care in both 2010 and 2011. RESULTS: Among 74,418 adults living with diagnosed HIV infection in the UK in 2010, 3510 (4.7%) were not reported as seen for clinical care or died in 2011. Case note reviews and outcomes were available for 2255 (64%) of these: 456 (20.2%) remained in the UK and did not attend care; 590 (26.2%) left UK; 508 (22.6%) received care in the UK: 73 (3.2%) died and 628 (27.8%) had no documented outcome. Individuals remaining in the UK and not attending care were more likely to be treatment naïve than those in care, but duration since HIV diagnosis was not significant. HIV/AIDS related hospitalisations were observed among non-attenders. CONCLUSION: Retention in UK specialist HIV care is excellent. Our audit indicates that the 'true' loss to follow up rate in 2011 was <2.5% with no evidence of health tourism. Novel interventions to ensure high levels of clinic engagement should be explored to minimise disease progression among non-attenders.

A Review of Plasmodium coatneyi-Macaque Models of Severe Malaria.

Malaria remains one of the most significant public health concerns in the world today. Approximately half the human population is at risk for infection, with children and pregnant women being most vulnerable. More than 90% of the total human malaria burden, which numbers in excess of 200 million annually, is due to Plasmodium falciparum. Lack of an effective vaccine and a dwindling stockpile of antimalarial drugs due to increased plasmodial resistance underscore the critical need for valid animal models. Plasmodium coatneyi was described in Southeast Asia 50 years ago. This plasmodium of nonhuman primates has been used sporadically as a model for severe malaria, as it mimics many of the pathophysiologic features of human disease. This review covers the reported macroscopic, microscopic, ultrastructural, and molecular pathology of P. coatneyi infection in macaques, specifically focusing on the rhesus macaque, as well as describing the critical needs still outstanding in the validation of this crucial model of human disease.

An overview of the HIV epidemic among men who have sex with men in the United Kingdom, 1999-2013.

We describe epidemiological trends in HIV among men who have sex with men (MSM) in the United Kingdom (UK) to inform prevention strategies. National HIV surveillance data were analysed for trends. Multivariable analyses identified predictors of late diagnosis (<350 copies/µL) and mortality. Between 1999 and 2013, 37,560 MSM (≥15 years) were diagnosed with HIV in the UK. New diagnoses rose annually from 1,440 in 1999 to 3,250 in 2013. The majority of MSM were of white ethnicity (85%) and UK-born (68%). Median CD4 count increased steadily from 350 cells/µL to 463 cells/µL. HIV testing in England increased from 10,900 tests in 1999 to 102,600 in 2013. One-year death rates after diagnosis declined among late presenters (4.7% to 1.9%). Despite declining late diagnosis (50% to 31%), the number of men diagnosed late annually has remained high since 2004. Older age (≥50 years), and living outside London were predictors of late presentation; older age and late presentation were predictors of one-year mortality. Increases in new diagnoses reflect increased testing and ongoing transmission. Over 900 men present late each year and mortality in this group remains high and preventable. Appropriate prevention and testing strategies require strengthening to reduce HIV transmission and late diagnosis.

HIV treatment as prevention among men who have sex with men in the UK: is transmission controlled by universal access to HIV treatment and care?

OBJECTIVES: In the UK, free HIV care is provided through dedicated HIV clinics. Using the national cohort of men who have sex with men (MSM) with diagnosed HIV infection and estimates of the number of undiagnosed men, we assessed whether high retention in HIV care and treatment coverage is sufficient to reduce HIV transmission. METHODS: Antiretroviral therapy (ART) uptake and viral load distribution among diagnosed men were analysed by treatment status and CD4 count for the period between 2006 and 2010. A multi-parameter evidence synthesis (MPES) method was used to estimate the size of the undiagnosed population. The viral load distribution among newly diagnosed untreated men was applied to the undiagnosed population. Infectivity was defined as a viral load > 1500 HIV-1 RNA copies/mL. RESULTS: Between 2006 and 2010, ART coverage among all HIV-infected MSM (diagnosed and undiagnosed) increased from 49 to 60%, while the proportion of infectious men fell from 47 to 35%. Over the same period, the number of all HIV-infected MSM increased from 30,000 to 40,100 and the number of infectious MSM remained stable at 14,000. Of the 14,000 infectious MSM in 2010, 62% were undiagnosed, 33% were diagnosed but untreated, and 5% received ART. Extending ART to all diagnosed HIV-infected MSM with CD4 counts < 500 cells/µL in 2010 would have reduced the overall proportion of infectious men from 35 to 29% and halving the proportion who were undiagnosed would further have reduced this to 21%. CONCLUSIONS: High ART coverage in the UK has reduced the infectivity of the HIV-diagnosed population. However, the effectiveness of treatment as prevention will be limited unless the undiagnosed population is reduced through frequent HIV testing and consistent condom use.

Quality of HIV care in the United Kingdom: key indicators for the first 12 months from HIV diagnosis.

OBJECTIVES: Prompt HIV diagnosis and treatment are associated with increased longevity and reduced transmission. The aim of the study was to examine late diagnoses and to assess the quality of care following diagnosis. METHODS: National surveillance and cohort data were used to examine late HIV diagnoses and to assess the quality of care received in the 12 months following HIV diagnosis. RESULTS: In 2011, 79% (4910/6219) of persons (15 years and over) diagnosed with HIV infection had CD4 counts reported within 3 months; of these, 49% were diagnosed late (CD4 count < 350 cells/µL). Adults aged 50 years and over were more likely to be diagnosed late (67%) compared with those aged 15-24 years (31%). Sixty-four per cent of heterosexual men were diagnosed late compared with 46% of women and 36% of men who have sex with men (MSM) (P < 0.01). The percentage of late diagnoses was highest among black African adults (66%) compared with other ethnicities; 96% of black African adults diagnosed late were born abroad. Overall, 88% and 97% of patients were linked to care within 1 and 3 months of diagnosis, respectively, with little variation by demographics and exposure category. The crude 1-year mortality rate was 31.6 per 1000 persons diagnosed in 2010. It was highest among adults diagnosed late (40.3/1000 versus 5.2/1000 for prompt diagnoses) and particularly among those aged 50 years and over. Excluding deaths, 85% of the 5833 diagnosed in 2010 were retained in care in 2011; 92% of the 2264 adults diagnosed late in 2010 received antiretroviral therapy by the end of 2011. CONCLUSIONS: The National Health Service provides high-quality care to persons newly diagnosed with HIV infection in the UK, with no evidence of health inequalities. Despite excellent care, half of adults are diagnosed late according to the threshold at which national guidelines recommend treatment should begin. Such patients have an 8-fold increased risk of 1-year mortality compared with those diagnosed promptly. Reducing late diagnosis of HIV infection remains a public health priority in the UK.

A maize line resistant to herbivory constitutively releases (E) -beta-caryophyllene.

Various pests, such as those in the order Lepidoptera, frequently feed on young maize (Zea mays) plants and pose a significant threat to plant development and survival. To manage this problem, maize generates a wide variety of responses to attack by pests, from activation of wound-response pathways to the release of volatile compounds. Mp708, an inbred line resistant to feeding by the larvae of the fall armyworm (Spodoptera frugiperda J.E. Smith Lepidoptera: Noctuidae), has been developed through traditional breeding methods, but its underlying mechanisms of resistance are still not completely understood. Mp708 has been shown to have a moderately high constitutive expression of jasmonic acid (JA) before infestation by fall armyworm. However, Tx601, a genotype susceptible to feeding by fall armyworm, activates JA pathway only in response to feeding, suggesting that Mp708 is "primed" to respond swiftly to an attack. Current research indicates that fall armyworm show a lack of preference to feeding on Mp708, leading to the hypothesis that volatiles constitutively released by the plant may also play an important role in its resistance. Analysis of volatiles released by Mp708 and Tx601 in the presence and absence of fall armyworm larvae identified (E)-beta-caryophyllene, a terpenoid associated with resistance, released constitutively in Mp708. Fall armyworm fed samples of both Mp708 and Tx601 showed high transcript number of tps23, the gene responsible for the synthesis of (E)-beta-caryophyllene. In addition, fall armyworm larvae show a preference for Tx601 whorl tissue over Mp708 tissue, and the dosage of Tx601 whorl with (E)-beta-caryophyllene repels the fall armyworm.

Restoring Carboxylates on Highly Modified Alginates Improves Gelation, Tissue Retention and Systemic Capture.

Alginate hydrogels are gaining traction for use in drug delivery, regenerative medicine, and as tissue engineered scaffolds due to their physiological gelation conditions, high tissue biocompatibility, and wide chemical versatility. Traditionally, alginate is decorated at the carboxyl group to carry drug payloads, peptides, or proteins. While low degrees of substitution do not cause noticeable mechanical changes, high degrees of substitution can cause significant losses to alginate properties including complete loss of calcium cross-linking. While most modifications used to decorate alginate deplete the carboxyl groups, we propose that alginate modifications that replenish the carboxyl groups could overcome the loss in gel integrity and mechanics. In this report, we demonstrate that restoring carboxyl groups during functionalization maintains calcium cross-links as well as hydrogel shear-thinning and self-healing properties. In addition, we demonstrate that alginate hydrogels modified to a high degree with azide modifications that restore the carboxyl groups have improved tissue retention at intramuscular injection sites and capture blood-circulating cyclooctynes better than alginate hydrogels modified with azide modifications that deplete the carboxyl groups. Taken together, alginate modifications that restore carboxyl groups could significantly improve alginate hydrogel mechanics for clinical applications. STATEMENT OF SIGNIFICANCE: Chemical modification of hydrogels provides a powerful tool to regulate cellular adhesion, immune response, and biocompatibility with local tissues. Alginate, due to its biocompatibility and easy chemical modification, is being explored for tissue engineering and drug delivery. Unfortunately, modifying alginate to a high degree of substitution consumes carboxyl group, which are necessary for ionic gelation, leading to poor hydrogel crosslinking. We introduce alginate modifications that restore the alginate's carboxyl groups. We demonstrate that modifications that reintroduce carboxyl groups restore gelation and improve gel mechanics and tissue retention. In addition to contributing to a basic science understanding of hydrogel properties, we anticipate our approach will be useful to create tissue engineered scaffolds and drug delivery platforms.

Travel for HIV care in England: a choice or a necessity?

OBJECTIVES: The aims of the study were (1) to measure the distance required to travel, and the distance actually travelled, to HIV services by HIV-infected adults, and (2) to calculate the proportion of patients who travelled beyond local services and identify socio-demographic and clinical predictors of use of non-local services. METHODS: The straight-line distance between a patient's residence and HIV services was determined for HIV-infected patients in England in 2007. 'Local services' were defined as the closest HIV service to a patient's residence and other services within an additional 5 km radius. Multivariable logistic regression was used to identify socio-demographic and clinical predictors of accessing non-local services. RESULTS: In 2007, nearly 57 000 adults with diagnosed HIV infection accessed HIV services in England; 42% lived in the most deprived areas. Overall, 81% of patients lived within 5 km of a service, and 8.7% used their closest HIV service. The median distance to the closest HIV service was 2.5 km [interquartile range (IQR) 1.5-4.2 km] and the median actual distance travelled was 4.8 km (IQR 2.5-9.7 km). A quarter of patients used a 'non-local' service. Patients living in the least deprived areas were twice as likely to use non-local services as those living in the most deprived areas [adjusted odds ratio (AOR) 2.16; 95% confidence interval (CI) 1.98-2.37]. Other predictors for accessing non-local services included living in an urban area (AOR 0.77; 95% CI 0.69-0.85) and being diagnosed more than 12 months (AOR 1.48; 95% CI 1.38-1.59). CONCLUSION: In England, 81% of HIV-infected patients live within 5 km of HIV services and a quarter of HIV-infected adults travel to non-local HIV services. Those living in deprived areas are less likely to travel to non-local services.

Prevalence of markers for HIV, hepatitis B and hepatitis C infection in UK military recruits.

An unlinked anonymous survey was conducted to measure the prevalence of selected markers for HIV, hepatitis B and C infection in recruits to the UK Armed Forces to inform future screening and hepatitis B vaccination policies. During 2007, nearly 14 000 left-over samples taken from new recruits for blood typing were collected, unlinked from identifiers and anonymously tested for HIV, hepatitis C and current and past cleared hepatitis B infection. Overall, serological evidence of HIV and hepatitis C was found in 0·06% and 0·06% of recruits, respectively. Evidence of past cleared and current hepatitis B infection was found in 3·63% and 0·37% of recruits, respectively. Overall, prevalence rates were broadly consistent with UK population estimates of infection. However, HIV and hepatitis B prevalence was higher in recruits of African origin than in those from the UK (P<0·0001). Screening for these infections is an option that could be considered for those entering Services from high-prevalence countries.

Implications for HIV testing policy derived from combining data on voluntary confidential testing with viral sequences and serological analyses.

OBJECTIVES: Laboratory, clinical and sequence-based data were combined to assess the differential uptake of voluntary confidential HIV testing (VCT) according to risk and explore the occurrence of HIV transmission from individuals with recently acquired HIV infection, before the diagnostic opportunity. METHODS: Between 1999 and 2002, nearly 30,000 anonymous tests for previously undiagnosed HIV infection were conducted among men who have sex with men (MSM) attending 15 sentinel sexually transmitted infection (STI) clinics in England, Wales and Northern Ireland. Using a serological testing algorithm, undiagnosed HIV-infected men were categorised into those with recent and non-recent infection. VCT uptake was compared between HIV-negative, recently HIV-infected and non-recently HIV-infected men. A phylogenetic analysis of HIV pol sequences from 127 recently HIV-infected MSM was conducted to identify instances in which transmission may have occurred before the diagnostic opportunity. RESULTS: HIV-negative MSM were more likely to receive VCT at clinic visits compared with undiagnosed HIV-infected MSM (56% (14,020/24,938) vs 31% (335/1072); p<0.001). Recently HIV-infected MSM were more likely to receive VCT compared with those with non-recent infections (42% (97/229) vs 28% (238/844); p<0.001). 22% (95/425) of undiagnosed HIV-infected MSM with STI received VCT. Phylogenetic analysis revealed at least seven transmissions may have been generated by recently HIV-infected MSM: a group that attended STI clinics soon after seroconversion. CONCLUSIONS: The integration of clinical, laboratory and sequence-based data reveals the need for specific targeting of the recently HIV exposed, and those with STI, for VCT. VCT promotion alone may be limited in its ability to prevent HIV transmission.

Cap Z(36/32), a barbed end actin-capping protein, is a component of the Z-line of skeletal muscle.

Various biological activities have been attributed to actin-capping proteins based on their in vitro effects on actin filaments. However, there is little direct evidence for their in vivo activities. In this paper, we show that Cap Z(36/32), a barbed end, actin-capping protein isolated from muscle (Casella, J. F., D. J. Maack, and S. Lin, 1986, J. Biol. Chem., 261:10915-10921) is localized to the barbed ends of actin filaments by electron microscopy and to the Z-line of chicken skeletal muscle by indirect immunofluorescence and electron microscopy. Since actin filaments associate with the Z-line at their barbed ends, these findings suggest that Cap Z(36/32) may play a role in regulating length, orienting, or attaching actin filaments to Z-discs.

Polyester fiber: from its invention to its present position.

The use of polyester fibers has grown more rapidly than that of any other man-made fiber. Many factors have contributed to this growth. Polyester's unique physical properties of strength, high modulus, elasticity, and durability are the basis for its success. The tailorability of the fiber makes it possible to generate a whole family of property variants for a wide variety of end uses. The ready availability and low cost of the raw materials, the continuing advances in polymerization technology, and the versatility of the melt spinning process have also been major factors in establishing polyester as the leading man-made fiber. New end uses have had a major impact on the growth of polyester. Most notable was the introduction of durable press fabrics for clothing and home furnishings. The consumer preference for easy-care fabrics and garments makes the durable press area one of continuing growth. Two relatively new areas where growth is expected to continue at a rapid rate are tire cord and textured knits for women's and men's outerwear.

The effect of in vitro treatment of bovine embryos with IGF-1 on subsequent development in utero to Day 14 of gestation.

Culture of bovine embryos with insulin-like growth factor-1 (IGF-1) can improve development to the blastocyst stage and embryo survival following transfer to heat-stressed, lactating dairy cows. Two experiments were conducted to determine whether IGF-1 could improve embryo survival and development at Day 14 after ovulation. In Experiment 1, non-lactating Holstein cows (n=58) were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced in vitro and cultured with or without 100ng/mL IGF-1. At Day 7 after expected ovulation (Day 0), groups of 7-12 embryos were randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and the presence or absence of an embryonic disc was recorded. Recovered embryos were cultured individually for 24h to determine interferon-tau (IFN-tau) secretion. There was no effect of IGF-1 on embryo recovery rate, embryo length or IFN-tau secretion. In Experiment 2, non-lactating (n=56) and lactating (n=35) Holstein cows were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced as described in Experiment 1. At Day 7 after expected ovulation (Day 0), a single embryo was randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and IFN-tau secretion were determined as in Experiment 1. Recovery rate at Day 14 tended (P=0.1) to be higher for recipients that received IGF-1 treated embryos compared to control embryos (43.2% versus 26.1%, respectively). There was no effect of IGF-1 on embryo length or IFN-tau secretion. In conclusion, results suggest that exposure to IGF-1 through Days 7-8 of development does not enhance capacity of embryos to prevent luteolysis. Results of the single embryo-transfer experiment suggested that IGF-1 treatment might affect embryo survival post-transfer as early as Day 14 after ovulation. Further experimentation is warranted to verify this finding.

Comparison of the metastatic properties of B16 melanoma clones isolated from cultured cell lines, subcutaneous tumors, and individual lung metastases.

Tumors produced by s.c. injection of uncloned B16 melanoma cell lines contain clonal tumor cell subpopulations with widely differing metastatic properties, including clones that are nonmetastatic. Similar metastatic heterogeneity exists in clones isolated from the same cell lines cultured in vitro. In B16 melanoma sublines (B16-BL6, B16-BV8, and B16-BP8) selected for enhanced invasive and metastatic behavior, the proportion of clones with high metastatic capacity is increased relative to the parent cell line. The cellular composition of metastases produced by s.c. or i.v. injection of the uncloned parent cell line has also been examined. Some metastases are populated by clones with indistinguishable metastatic properties (intralesional clonal homogeneity) while others yield clones with different metastatic properties (intralesional clonal heterogeneity). The range of clonal diversity in heterogeneous metastases is, however, substantially less than in the parent line. The number of metastases yielding clones with heterogeneous metastatic phenotypes is higher for "spontaneous" metastases arising from s.c. tumors than in "experimental" metastases produced by i.v. injection of single-cell suspensions. Studies using B16 cells bearing specific biochemical markers indicate that clonally homogeneous metastases are of monoclonal origin and that metastases populated by clones with heterogeneous metastatic phenotypes are of polyclonal origin.

Subfractionation of liver membrane preparations by specific ligand-induced density perturbation.

Immunofluorescence and immunoferritin staining with monospecific antibodies to dipeptidyl peptidase IV purified from rat liver plasma membrane showed that the antigenic sites of this glycoprotein was exposed only on the outer surface of the liver cell. In a vesiculated plasma membrane preparation the peptidase was located exclusively on right-side-out elements, which differed in their degrees of ferritin staining, and could be separated into subfractions of different buoyant densities corresponding to their concentration of dipeptidyl peptidase IV. The concomitant density perturbation of nucleotide pyrophosphatase was similar, but not identical, to that of the peptidase itself, indicating that these two marker enzymes are somewhat differently distributed in the plane of the liver plasma membrane. Since essentially all the galactosyl transferase in plasma membrane and none of that in Golgi membrane could be density-perturbed with the antipeptidase, the activity in the plasma membrane preparation could not be ascribed to contamination with discrete Golgi elements. On the other hand, the small amount of dipeptidyl peptidase IV found in the Golgi preparations was itself perturbed by the antipeptidase, indicating that it represented contaminating right-side-out plasma membrane vesicles. In preliminary experiments similar separations were also obtained with wheat germ agglutinin as the plasma membrane ligand. Density perturbation, mediated by the recognition of specific surface markers, should be a useful adjunct in the separation and characterization of subcellular components in other systems.

Improved method for the isolation of rat liver plasma membrane.

An improved method for the isolation of plasma membrane from rat liver is presented. Gentle homogenization of perfused livers in buffered isotonic KCL, followed by direct flotation of a low-speed nuclear pellet through a discontinuous sucrose density gradient results in a 32% yield, and 25-fold enrichment for the plasma membrane marker, phosphodiesterase I, in a crude plasma membrane fraction. This fraction contains less than 1% of the mitochondria, and endoplasmic reticulum present in the original homogenate, but is more heavily contaminated with lysosomes and Golgi membrane. Vigorous mechanical disruption of this material, followed by a second discontinuous sucrose density gradient, gives a light plasma membrane fraction with an 80-fold purification and 20% yield of phosphodiesterase I over the original homogete (with further reduction of contaminants).