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The Julia Creek dunnart, Sminthopsis douglasi , is a small, threatened carnivorous marsupial occurring in scattered populations in the grasslands of central and northwestern Queensland, Australia. The distribution of the species is largely unknown due to sporadic survey efforts and its historically low detection using traditional live trapping methods. There is an urgent need to determine the best methods of detection to optimise survey methodologies and more effectively manage species conservation efforts. In this study, we compared the effectiveness of live (Elliott) traps, baited white flash camera traps and thermal imagery binocular surveying for detecting S. douglasi . We deployed 40 white flash camera traps at two sites in Bladensburg National Park (south of Winton), where the species is known to occur, for three consecutive periods between June and November 2022. Four comparative sessions of live trapping were undertaken between April and August 2022 at the same locations. During the live trapping periods, a total of 12 nights of surveying were conducted with thermal imagery binoculars in a preliminary assessment of the technique. The total live trapping effort was 3600 trap nights (approximately 700 trap nights per site in each trapping event). Live trapping resulted in 12 detections of individual S. douglasi from 19 total captures. The highest trap success on a given trapping session was 1.71%, and overall trap success from both sites across all sessions was 0.53%. In comparison, baited camera traps (deployed facing the ground at 70 cm range) took 1,269,884 images over 5383 trap nights. There were 11 confirmed images of S. douglasi , on three individual occasions, which represented 2.10% of all small mammal captures and just 0.0009% of the total images. Four species of small mammals were detected using camera traps, whereas live trapping detected only two species. No small mammals were detected on any of the 12 thermal binocular surveys. Overall, our study highlights the comparative high utility of traditional live trapping for detecting S. douglasi . This research provides a framework for ongoing monitoring of the Bladensburg National Park population. It will be more broadly beneficial for informing the best detection techniques of S. douglasi in ongoing work investigating the overall distribution of the species. Similar studies assessing multiple detection methods for small terrestrial mammals have shown an advantage of white flash camera traps compared to other traditional detection techniques. Our contrasting results serve as a reminder that the utility of different techniques for detecting small mammals is best assessed on a species-by-species basis., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

Globally, hundreds of mammal species face the threat of extinction in the coming decades, and in many cases, their ecology remains poorly understood. Fundamental ecological knowledge is crucial for effective conservation management of these species, but it is particularly lacking for small, cryptic mammals. The Julia Creek dunnart ( Sminthopsis douglasi ), a threatened, cryptic carnivorous marsupial that occurs in scattered populations in the central west of Queensland, Australia, was once so poorly studied that it was believed extinct. Sporadic research since its rediscovery in the early 1990s has revealed that S. douglasi is distributed across land at risk from many threats. Fundamental knowledge of S. douglasi population density is urgently required to inform conservation management at key sites, yet the species has historically proven hard to detect. Indeed, the status of the largest known population of S. douglasi , in Bladensburg National Park, is unknown. Here, we conducted a population study on S. douglasi at two sites within Bladensburg National Park via live mark-recapture surveys during 2022 and 2023. From likelihood-based spatially explicit capture-recapture (SECR) modelling we provide the first estimates of density and population size for S. douglasi . Live trapping resulted in captures of 49 individual S. douglasi (with 83 captures total, including recaptures). We estimated S. douglasi to occur at a density of 0.38 individuals ha -1 (0.25-0.58) at one site and 0.16 individuals ha -1 (0.09-0.27) at another site, with an estimated mean population size in suitable habitat at Bladensburg National Park of 1211 individuals (776-1646). Our S. douglasi density estimates were similar to that reported for other threatened small mammals in Australia. We also found evidence of extreme S. douglasi population fluctuations over time at Bladensburg National Park, which is of concern for its future conservation. Our study has provided the first estimate of density for S. douglasi , a threatened dasyurid species from the Mitchell Grass Downs of central western Queensland, Australia. Our research provides crucial population data to assist the management of this poorly studied species. We demonstrate a method that can be applied to species with low detection probability to ultimately help address the mammal extinction crisis faced by Australia and the rest of the world., Competing Interests: The authors declare that there are no competing interests., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

Background: Selection committees try to ascertain that motivated students are selected for medical school. Self-determination theory stresses that the type of motivation is more important than the quantity of motivation. Autonomous motivation, compared to controlled motivation, in students leads to better learning outcomes. Applicants can express their motivation in written statements, a selection tool which has been found to elicit heterogeneous responses, hampering the comparison of applicants. This study investigates the content of applicants' statements on motivation for medical school in particular, the possibility to distinguish the type of motivation and the differences between selected and non-selected applicants., Methods: A thematic analysis was conducted on written statements on motivation (n = 96), collected as a part of the selection procedure for the graduate entry program for medicine and research at our institution. Themes were identified as motivation-related and motivation-unrelated (additional). The motivation-related themes were further classified as autonomous and controlled types of motivation. Group percentages for each theme were compared between selected and non-selected applicants using Chi-square test and Fisher exact test., Results: Applicants mainly described reasons belonging to autonomous type of motivation and fewer reasons belonging to controlled type of motivation. Additional themes in the statements included previous work experience and academic qualifications, ambitions, expectations and descriptions of the program and profession, personal qualities, and personal history. Applicants used strong words to support their stories. The selected and non-selected applicants did not differ in their types of motivation. Non-selected applicants provided more descriptions of personal history than selected applicants (p < 0.05)., Conclusions: The statement on motivation does not appear to distinguish between applicants in selection for medical school. Both selected and non-selected applicants reported mainly autonomous motivation for applying, and included a lot of additional information, which was beyond the scope of what was asked from them. The findings raise a question mark on the validity and reliability of the statement on motivation as a tool for selection. It could however be of added value to enable applicants to tell their story, which they appreciate, and to create awareness of the program, resulting in an informed decision to apply.

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.

Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing beta-cells in the pancreas. In this process, islet epitope-specific CD8(+) T-cells play a pivotal role. Thus, monitoring of multiple islet-specific CD8(+) T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive., Research Design and Methods: Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B(10-18), prepro-insulin (PPI)(15-24), islet antigen (IA)-2(797-805), GAD65(114-123), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(265-273), and prepro islet amyloid polypeptide (ppIAPP)(5-13)-specific CD8(+) T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients., Results: Using this kit, islet autoreactive CD8(+) T-cells recognizing insulin B(10-18), IA-2(797-805), and IGRP(265-273) were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI(15-24) proved to be the most sensitive epitope. Applying the "Diab-Q-kit" to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell-derived epitopes that were associated with disease activity and correlated with clinical outcome., Conclusions: A kit was developed that allows simultaneous detection of CD8(+) T-cells reactive to multiple HLA-A2-restricted beta-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.

The use of fluorescently labeled major histocompatibility complex multimers has become an essential technique for analyzing disease- and therapy-induced T-cell immunity. Whereas classical major histocompatibility complex multimer analyses are well-suited for the detection of immune responses to a few epitopes, limitations on human-subject sample size preclude a comprehensive analysis of T-cell immunity. To address this issue, we developed a combinatorial encoding strategy that allows the parallel detection of a multitude of different T-cell populations in a single sample. Detection of T cells from peripheral blood by combinatorial encoding is as efficient as detection with conventionally labeled multimers but results in a substantially increased sensitivity and, most notably, allows comprehensive screens to be performed. We obtained proof of principle for the feasibility of large-scale screening of human material by analysis of human leukocyte antigen A3-restricted T-cell responses to known and potential melanoma-associated antigens in peripheral blood from individuals with melanoma.

Recombinant major histocompatibility complex (MHC) class I molecules complexed with pathogen-specific or other disease-associated antigens have become essential reagents for the analysis of adaptive T-cell responses. However, conventional techniques for the production of recombinant peptide-MHC (pMHC) complexes are highly involved and thereby limit the use of pMHC complexes in terms of antigen diversity. To make pMHC-based techniques suitable for high-throughput analyses we developed an MHC peptide exchange technology based on the use of conditional MHC ligands. This technology enables the parallel production of thousands of different pMHC complexes within hours, allowing the development of high-throughput MHC-based assay systems to identify MHC ligands and cytotoxic T-cell responses. These high-throughput assays should prove valuable for the screening of entire disease-associated proteomes, including pathogen-encoded proteomes, tumor-associated antigens, and autoimmune antigens.

Objective: To obtain insight in the extent to which the human cell lines LiSa-2 and PAZ6 resemble isolated primary human adipocytes., Design: A combination of cDNA subtraction (representative difference analysis; RDA) and cDNA microarray analysis was used to select adipose specific genes to compare isolated (pre-)adipocytes with (un)differentiated LiSa-2 and PAZ6 cells., Measurements: RDA was performed on adipose tissue against lung tissue. A total of 1400 isolated genes were sequenced and cDNA microarray technology was used for further adipose related gene selection. 30 genes that were found to be enriched in adipose tissue were used to compare isolated human adipocytes and LiSa-2 and PAZ6 cells in the differentiated and undifferentiated states., Results: RDA and microarray analysis resulted in the identification of adipose enriched genes, but not in adipose specific genes. Of the 30 most differentially expressed genes, as expected, most were related to lipid metabolism. The second category consisted of methyltransferases, DNMT1, DNMT3a, RNMT and SHMT2, of which the expression was differentiation dependent and higher in differentiated adipocytes. Using the 30 adipose expressed genes, it was found that isolated adipocytes on one hand, and PAZ6 and LiSa-2 adipocytes on the other, differ primarily in lipid metabolism. Furthermore, LiSa-2 cells seem to be more similar to isolated adipocytes than PAZ6 cells., Conclusion: The LiSa-2 cell line is a good model for differentiated adipocytes, although one should keep in mind that the lipid metabolism in these cells deviates from the in vivo situation Furthermore, our results imply that methylation may have an important function in terminal adipocyte differentiation.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

PPARgamma is obligatory for fat mass generation and is thought to determine the amount of TAG stored per fat cell. We investigated whether ligand availability for PPARgamma is rate limiting in fat mass generation and substrate metabolism. Twenty healthy men (20-29 years) were randomly assigned to receive the PPARgamma ligand rosiglitazone (RSG) (8 mg/d) (n 10) or a placebo (n 10) during a stay of 7 d in a respiration chamber. Food intake was ad libitum, resulting in positive energy balances of 32.2 MJ (placebo) and 44.7 MJ (RSG). Fat cell size and expression of PPARgamma, adipocyte fatty acid-binding protein (aP2), adipsin, adiponectin and fasting-induced adipose factor (FIAF) were determined in subcutaneous abdominal fat biopsies. The total amount of fat stored and the amount of TAG per fat cell were not different between groups. For the entire group, fat cell size was decreased after overeating (P = 0.02). FIAF mRNA levels were decreased after overeating in the RSG group (P = 0.01), with a trend towards a decrease in the placebo group. Unexpectedly, RSG treatment did not influence the expression levels of PPARgamma and of the PPARgamma responsive genes aP2, adiponectin and adipsin. In addition, RSG resulted in a larger increase in plasma TAG during overeating than placebo treatment. These results suggest that in healthy, non-obese males the PPARgamma ligand RSG influences TAG metabolism, independent of its PPARgamma transcriptional activity in the subcutaneous adipose tissue.

Adipose tissue is a highly plastic tissue with an important endocrine and metabolic function. To understand its role in human health and disease, it is necessary to understand the extent of variation and the specific differences within and between different depots and subjects. We employed cDNA microarray analysis to investigate this in human subjects ranging from lean to mildly obese. We observe (1) high similarity between different samples of one adipose depot, (2) only small differences between the subcutaneous and visceral adipose tissue depot and (3) larger differences in gene expression between different individuals (per depot). The major variation within adipose depots can be attributed to differences in the non-adipocyte component of adipose tissue. Using only non-obese subjects, we identified genes that were consistently differentially expressed between subcutaneous and omental adipose tissue, despite the variation in gene expression between these subjects. Using quantitative real time polymerase chain reaction (PCR), fatty acid binding protein 4 (FABP4), vimentin (Vim), four and a half LIMs domains (FHL1), CD36 (all higher in subcutaneous adipose tissue) and Matrix Gla protein (MGP; lower in subcutaneous adipose tissue) were confirmed to be significantly differentially expressed between depots.

Objective: This study investigates whether fasting plasma glucose (FPG) levels in obese subjects are associated with the number of preadipocytes and their adipokine-secretion capabilities., Design: Abdominal subcutaneous and omental adipose tissues were obtained from 10 female and four male obese subjects (age 37 +/- 8 years; BMI 48 +/- 13 kgm(2)) with a wide range of FPG (range: 4.3-10.6 mm). Stromal vascular cells (SVC) were isolated and cultured and the number of attached SVC (aSVC) per gram adipose tissue determined. The aSVCs were differentiated in vitro to become adipocytes, and the secretion of the adipokine leptin and adiponectin in the culture media was determined. Spearman rank correlation coefficients were calculated between FPG and preadipocyte number and adipokine secretion., Patients: Subject-inclusion criteria: BMI >40 kg/m(2) and for severe comorbid conditions BMI >35 kg/m(2). Subject-exclusion criteria: severe cardiopulmonary pathology (ASA class 3), history of bariatric surgery, manifest psychopathology, 18 years < age > 60 years and for upper-abdominal surgery, age >50 years. All females in the study had regular menstrual periods. None of participants received glucose-lowering medication., Results: No association was observed between BMI and fasting glucose levels. More than 90 +/- 20% of the cultured aSVC fraction was able to store fat droplets, indicating the presence of preadipocytes. A strong negative association was observed between omental preadipocyte number and FPG. A strong association was observed between adipokine secretion by the omental preadipocytes and FPG. No association was observed between subcutaneous preadipocyte number and adipokine secretion and FPG., Conclusions: In morbid obese subjects, low number of omental preadipocytes with high-leptin- and low-adiponectin-secretion profiles is associated with high FPG.

Both apolipoprotein E (apoE) and 24(S)-hydroxycholesterol are involved in the pathogenesis of Alzheimer disease (AD). It has been hypothesized that apoE affects AD development via isoform-specific effects on lipid trafficking between astrocytes and neurons. However, the regulation of the cholesterol supply of neurons via apoE-containing high density lipoproteins remains to be clarified. We show for the first time that the brain-specific metabolite of cholesterol produced by neurons, i.e. 24(S)-hydroxycholesterol, induces apoE transcription, protein synthesis, and secretion in a dose- and time-dependent manner in cells of astrocytic but not of neuronal origin. Moreover, 24(S)-hydroxycholesterol primes astrocytoma, but not neuroblastoma cells, to mediate cholesterol efflux to apoE. Similar results were obtained using the synthetic liver X receptor (LXR) agonist GW683965A, suggesting involvement of an LXR-controlled signaling pathway. A 10-20-fold higher basal LXRalpha and -beta expression level in astrocytoma compared with neuroblastoma cells may underlie these differential effects. Furthermore, apoE-mediated cholesterol efflux from astrocytoma cells may be controlled by the ATP binding cassette transporters ABCA1 and ABCG1, since their expression was also up-regulated by both compounds. In contrast, ABCG4 seems not to be involved, because its expression was induced only in neuronal cells. The expression of sterol regulatory element-binding protein (SREBP-2), low density lipoprotein receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and SREBP-1c was transiently up-regulated by GW683965A in astrocytes but down-regulated by 24(S)-hydroxycholesterol, suggesting that cholesterol efflux and synthesis are regulated independently. In conclusion, evidence is provided that 24(S)-hydroxycholesterol induces apoE-mediated efflux of cholesterol in astrocytes via an LXR-controlled pathway, which may be relevant for chronic and acute neurological diseases.

Background and Aim: Fat mass generation requires an energy surplus and the activity of the peroxisome proliferator-activated receptor gamma (PPARgamma). We investigated if the PPARgamma ligand rosiglitazone influences substrate usage, energy expenditure (EE) and energy intake (EI) and, thereby, how PPARgamma activity contributes to susceptibility to obesity., Methods: Twenty healthy males (20-29 years) were randomly assigned to receive a placebo (n = 10) or rosiglitazone (8 mg/d) (n = 10) for seven consecutive days, while staying in a respiration chamber. Food intake was ad libitum. Body composition was determined by underwater weighing (day 1) and deuterium dilution (day 1 and 8)., Results: Mean (+/-SE) EI was 15.9 +/- 0.9 MJ/d in the placebo group and 18.9 +/- 1.2 MJ/d in the rosiglitazone group. Mean EE was 11.3 +/- 0.3 MJ/d and 12.5 +/- 0.5 MJ/d for the placebo and rosiglitazone groups respectively. This resulted in a cumulative positive energy balance (EB) of 32.3 +/- 5.1 MJ for placebo and 44.7 +/- 6.9 MJ for rosiglitazone. There were no significant differences in EI, EE, and EB between treatments. Both groups did not adjust their fat oxidation to the increased fat intake, but fat oxidation decreased faster in the rosiglitazone group (significantly lower on days 6 and 7). During treatment with rosiglitazone, significantly more fat storage was seen in overweight subjects while this was not the case in the placebo group., Conclusions: Our results suggest a shift in substrate usage during PPARgamma stimulation leading to a preference for fat storage, especially in subjects with a higher BMI.

Objective: As the peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in fat mass regulation, we investigated whether initial subcutaneous PPARgamma activity is related to fat mass generation during overfeeding., Subjects: Fourteen healthy female subjects (age 25 +/- 4 years, BMI 22.1 +/- 2.3 kg/m2)., Design and Measurements: Subjects were overfed with a diet supplying 50% more energy than baseline energy requirements for 14 days. Fasting blood samples were analyzed for leptin, insulin and glucose. Fasting subcutaneous abdominal fat biopsies were obtained for analysis of PPARgamma1, PPARgamma2, aP2 and UCP2 mRNAs., Results: Initial PPARgamma1 and 2, aP2 and UCP2 mRNAs were not related to fat gain (P > 0.12). However, PPARgamma1, PPARgamma2 and aP2 mRNA changes were positively related to changes in plasma leptin (P < 0.05) and, except aP2 (P = 0.06), to fat gain (P < 0.05). PPARgamma and aP2 mRNA changes were positively related (P<0.01), indicating that PPARgamma mRNA levels reflected PPARgamma activity., Conclusion: These data suggest that the ability to increase PPARgamma activity might be involved in the susceptibility to gain weight during a positive energy balance.

Objective: To investigate whether efficiency of weight gain during a short period of overfeeding is related to adaptive differences in basal metabolic rate (BMR) and physical activity., Subjects: Fourteen healthy females (age 25+/-4 years, BMI 22.1+/-2.3 kg/m2)., Design and Measurements: Subjects were overfed with a diet supplying 50% more energy than baseline energy requirements for 14 days. Overfeeding diets provided 7% of energy from protein, 40% from fat and 53% from carbohydrates. Body composition was determined using hydrodensitometry and isotope dilution, total energy expenditure (TEE) with doubly labeled water and basal metabolic rate (BMR) with indirect calorimetry. Physical activity (PA) was recorded with a tri-axial accelerometer., Results: Body weight increased by 1.45+/-0.86 kg (mean+/-S.D.) (P<0.0001), fat mass increased by 1.05+/-0.75 kg. Energy storage was 57.0+/-17.9 MJ, which is the difference between energy intake (207.2 MJ) and energy expenditure (150.2 MJ) during overfeeding. There was no difference between metabolically efficient and metabolically inefficient subjects in changes in BMR and PA., Conclusion: These results indicate that the metabolic efficiency of weight gain was not related to adaptive changes in energy expenditure.

The detection of antigen-specific T cell responses by MHC multimer staining is rapidly becoming one of the core immunological techniques, and the technology to produce MHC multimers has been optimized substantially in recent years. Furthermore, recent work demonstrates the potential of high-throughput detection of T cell responses and suggests that manipulation of T cell responses through the use of multimeric MHC reagents is also feasible.

Activated CD8+ T cells detect virally infected cells and tumor cells by recognition of major histocompatibility complex class I-bound peptides derived from degraded, endogenously produced proteins. In contrast, CD8+ T cell activation often occurs through interaction with specialized antigen-presenting cells displaying peptides acquired from an exogenous cellular source, a process termed cross-priming. Here, we observed a marked inefficiency in exogenous presentation of epitopes derived from signal sequences in mouse models. These data indicate that certain virus- and tumor-associated antigens may not be detected by CD8+ T cells because of impaired cross-priming. Such differences in the ability to cross-present antigens should form important considerations in vaccine design.

Objective: To determine the variation in preadipocyte isolation procedure and to assess the number and function of preadipocytes from subcutaneous and omental adipose tissue of obese individuals., Research Methods and Procedures: The preadipocyte number per gram of adipose tissue in the abdominal-subcutaneous and abdominal-omental adipose stores of 27 obese subjects with a BMI of 44 +/- 10 kg/m(2) and an age of 40 +/- 9 years was determined., Results: The assessment of the preadipocyte number was found to be labor intensive and error prone. Our data indicated that the number of stromal vascular cells (SVCs), isolated from the adipose tissue by collagenase digestion, was dependent on the duration of collagenase treatment and the size and the origin of the biopsy. In addition, the fat accumulation and leptin production by differentiated SVCs were dependent on the number of adherent SVCs (aSVCs) in the culture plate and the presence of proteins derived from serum and peroxisome proliferator-activated receptor ligands., Discussion: Using our standardized isolation and differentiation protocol, we found that the number of SVCs, aSVCs, leptin production, and fat accumulation still varied considerably among individuals. Interestingly, within individuals, the number of SVCs, aSVCs, and the leptin production by differentiating aSVCs from both the subcutaneous and the omental fat depots were associated, whereas fat accumulation was not. In obese to severely obese subjects, differences in BMI and age could not explain differences in SVCs, aSVCs, leptin production, and fat accumulation.

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

Antigen presentation by MHC class II molecules relies on the action of endocytic proteases, which are differentially expressed in antigen-presenting cells and are regulated by different components of the immune system. Endocytic enzymes process and convert exogenous antigens into peptidic determinants capable of interaction with MHC class II molecules. Chemical and genetic tools have recently been developed to study the role of lysosomal proteases in antigen presentation.

Summary: The present study provides evidence that chronic intake of a high-fat diet induces a dramatic extravasation of immunoglobulins, indicating alterations in blood-brain barrier (BBB) functioning, in the brains of apolipoprotein E (apoE)-knockout mice, but not of C57Bl/6 control mice. Using sodium fluorescein as a marker for the permeability of the BBB, we found additional support for age-related disturbances of BBB function in apoE-knockout mice. Behavioral analysis of apoE-knockout mice compared with C57Bl/6 mice indicated that they were also less efficient in acquiring the spatial Morris water maze task. Furthermore, apoE-knockout mice are known to develop severe atherosclerosis, which is exacerbated with a high-fat diet. We therefore compared the apoE-knockout mice with the apoE3-Leiden transgenic mice, which are known to develop atherosclerosis. However, apoE3-Leiden mice that were kept on a high-fat, high-cholesterol diet and that developed atherosclerosis to an extent similar to the apoE-knockout mice, showed no signs of BBB disturbances. These results indicate for the first time that apoE plays an essential role in the maintenance of the integrity of the BBB during aging and that it protects the brain from neuropathology induced by a high-fat diet. We therefore hypothesize that the role of apoE in the maintenance of the integrity of the BBB may be the mechanism by which apoE affects the progression of neurodegeneration, as seen in Alzheimer's disease.

Objective: To test the hypothesis that consumption of a high-fat diet leads to an increase in UCP mRNA expression in human skeletal muscle. In a group of endurance athletes, with a range in fiber type distribution, we hypothesized that the effect of the high-fat diet on UCP2 and UCP3 mRNA expression is more pronounced in muscle fibers which are known to have a high capacity to shift from carbohydrate to fat oxidation (type IIA fibers)., Design: Ten healthy trained athletes (five males, five females) consumed a low-fat diet (17+/-0.9 en% of fat) and high-fat diet (41.4+/-1.4 en% fat) for 4 weeks, separated by a 4 week wash-out period. Muscle biopsies were collected at the end of both dietary periods., Measurements: Using RT-PCR, levels of UCP2 and UCP3 mRNA expression were measured and the percentage of type I, IIA and IIB fibers were determined using the myofibrillar ATPase method in all subjects., Results: UCP3L mRNA expression tended to be higher on the high-fat diet, an effect which reached significance when only males were considered (P=0.037). Furthermore, diet-induced change in mRNA expression of UCP3T (r: 0.66, P=0.037), UCP3L (r: 0.61, P=0.06) and UCP2 (r: 0.70, P=0.025), but not UCP3S, correlated significantly with percentage dietary fat on the high-fat diet. Plasma FFA levels were not different during the two diets. Finally, the percentage of type IIA fibers was positively correlated with the diet-induced change in mRNA expression for UCP2 (r: 0.7, P=0.03), UCP3L (r: 0.73, P=0.016) and UCP3T (r: 0.68, P=0.03) but not with UCP3S (r: 0.06, NS)., Conclusion: UCP2 and UCP3 mRNAs are upregulated by a high-fat diet. This upregulation is more pronounced in humans with high proportions of type IIA fibers, suggesting a role for UCPs in lipid utilization.

Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.

We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.

Objective: To determine whether physical disability, pain, depressive mood, and criticism by the spouse are differentially related to intrusiveness of rheumatoid arthritis (RA) on sexuality in male and female patients., Methods: Physical and psychological aspects of health were assessed in 102 male and 118 female RA patients who were living with a spouse. Patients were classified into 3 levels of intrusiveness of RA on sexuality. The data were analyzed by means of analysis of covariance and multiple regression analysis., Results: Greater intrusiveness of RA on sexuality was related to greater physical disability, pain, and depression in male and female RA patients. Female patients, compared with male patients, appeared to have lower levels of mobility and self-care. Male and female patients did not differ in their level of intrusiveness of RA on sexuality., Conclusion: Physical disability, pain, and, to a lesser extent, depression were found to contribute to intrusiveness of RA on sexuality. It is suggested that differences in sexual motivation between men and women might have been influential in the absence of gender differences in intrusiveness.

Fibrin-dependent plasminogen activation by tissue-type plasminogen activator (t-PA) is in part associated with the presence of the kringle 2 domain in t-PA. Within this kringle 2 domain a lysyl-binding site has been described. The plasminogen to plasmin conversion by urokinase-type plasminogen activator (u-PA), in contrast to that of t-PA, is not enhanced in the presence of fibrin. Within the u-PA kringle domain no lysyl-binding site is found. To study whether introduction of a lysyl-binding site in the u-PA kringle domain will make u-PA a fibrin-dependent plasminogen activator, three stretches of amino acid residues of the u-PA kringle domain (A28-Q33, D55-N57 and G67-V72) were substituted by three stretches of amino acids from the corresponding positions of the kringle 2 domain of t-PA (M28-K33, D55-D57 and N67-W72). These changes resulted in the creation of the lysyl-binding site consensus of the kringle 2 domain (K33, D55, D57, W62 and W72) in the u-PA kringle. However, the resulting u-PA mutant did not interact with lysyl-Sepharose, nor did it display fibrin-enhanced plasminogen activation in the presence of soluble fibrin mimic. When the kringle domain of u-PA was replaced by the kringle 2 domain of t-PA, similar results were obtained. The hybrid protein hardly interacted with lysyl-Sepharose and the plasminogen activation was not enhanced in the presence of fibrin mimic. However, the N-terminal fragment isolated from this hybrid molecule (consisting of growth factor domain and kringle 2 domain) did interact with lysyl-Sepharose, suggesting that in the hybrid molecule a functional lysyl-binding site is present but not operational. Indeed, lysine analogue (epsilon-amino-caproic acid) sensitive binding of isolated t-PA kringle 2 domain to u-PA could be observed. The modified u-PA kringle, the wild type u-PA kringle and the kringle 2 of the u-PA hybrid were also placed N-terminal of the protease domain of t-PA. As expected, the t-PA mutant consisting of the kringle 2 domain and the protease domain bound to lysyl-Sepharose and showed fibrin-dependent plasminogen activation. Further, the hybrid molecule consisting of the u-PA kringle placed N-terminal of the t-PA protease domain did not display these features. Introduction of the modified u-PA kringle N-terminal of the t-PA protease domain resulted in a very weak interaction with lysyl-Sepharose. Despite the high overall similarity in primary structure of the modified u-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogen activation of this hybrid molecule was observed. The above-mentioned results question the concept that the structural auto-nomous domains within hybrid plasminogen activators t-PA and u-PA function as autonomous domains and suggest that interactions between the kringle and the protease domain in hybrid molecules strongly influences their functional features.

To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the absence and the presence of epsilon-amino caproic acid (EACA). Occupation of the lysyl binding site in the K2 domain with EACA has a pronounced effect on the binding of FGK1K2P to a fibrin clot (C50 = 77 +/- 11 nM versus 376 +/- 45 nM with EACA). Deleting the lysyl binding site in the K2 domain (substitution D236N) also impairs fibrin binding but to a lesser extent (C50 = 169 +/- 20 nM). Although the binding of K2P to a fibrin clot is weak (C50 = 1163 +/- 490 nM), it still is 2 orders of magnitude stronger than the binding of EACA to K2P. Therefore it was surprising to find that deletion of the lysyl binding site in K2P completely abolishes fibrin binding. Even when both the F domain and the lysyl binding site were deleted, considerable fibrin binding is still observed (C50 = 557 +/- 126 nM), suggesting other than F and K2-mediated interactions. The binding of FGK1K2P, FGK1K2P (D236N), GK1K2P, and GK1K2P (D236N) to fibrin could be competitively inhibited by FGK1K2P and K2P, indicating that all molecules recognize the same interaction sites on a fibrin clot. Based on these results, a new model for the interaction of t-PA with a forming fibrin clot is proposed. The fibrin binding sites in t-PA are not confined to the F and K2 domain. The main role of the lysyl binding site in the K2 domain of t-PA appears indirect rather than direct, most likely stabilizing a conformation favorable for fibrin binding.

Tissue-type plasminogen activator (t-PA) is composed of structurally autonomous domains. From the N-terminus of t-PA, a finger-like domain (F), an epidermal growth factor-like domain (G), two kringle domains (K1 and K2) and a serine protease domain (P) can be discerned. The K2 domain of t-PA is known to be involved in lysine binding, fibrin binding and fibrin-dependent plasminogen activation. To study the functional autonomy of the K2 domain in t-PA we constructed, with the aid of a cassette t-PA gene [Rehberg et al. (1989) Protein Engng, 2, 371-377], mutant t-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2P and GK2K1P) in which the K2 domain was placed in two different positions in t-PA. The DNAs of wild-type t-PA and the t-PA variants were expressed in Chinese hamster ovary cells and the recombinant proteins were purified by affinity chromatography. All molecules were expressed in their single-chain form and could be converted to their two-chain form. With these molecules, lysine binding, fibrin binding and fibrin-dependent plasminogen activation were studied. All variants showed affinity for lysyl-Sepharose and aminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versus FGK2K1P and GK1K2P versus GK2K1P) resulted in a 23-47% weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose. Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versus GK2K1P) resulted in a 20-70% improvement of the interactions lysyl-Sepharose and aminohexyl-Sepharose.(ABSTRACT TRUNCATED AT 250 WORDS)

Despite the high overall similarity in primary structure between kringle one (K1) and kringle two (K2) of tissue-type plasminogen activator (t-PA) there exists an enormous functional difference. It is thought that, in contrast to K1, K2 mediates lysine binding and fibrin binding and is involved in stimulation of plasminogen activation by fibrin or derivatives as CNBr fragments of fibrinogen. Hypothesizing that sequence differences are responsible for differences in function, we compared the amino acid sequences of K1 and K2 with a consensus kringle sequence. Six consecutive amino acids unique to K2 of t-PA were found, i.e. from Asn248 to Trp253. To test whether these residues are involved in lysine binding, fibrin binding, and fibrin-dependent plasminogen activation, we constructed a set of t-PA mutant proteins containing only a kringle and the protease (P) domain: K2P, K1P, and k1P. In the latter molecule the original amino acid residues Ala160-Ser165 from K1 were substituted by Asn248-Trp253 from K2. As expected, K2P showed enhanced plasminogen activation in the presence of CNBr fragments of fibrinogen, bound to lysine-Sepharose and to a forming fibrin clot. K1P did not show any of these features. In contrast, k1P could be stimulated by CNBr fragments of fibrinogen and bound to lysine-Sepharose and a forming fibrin clot. These results indicate that at least a part of the functional differences between K1 and K2 of t-PA can be localized to a stretch of 6 amino acid residues from Asn248 to Trp253 present in K2.