Projecting boreal bird responses to climate change: the signal exceeds the noise.

For climate change projections to be useful, the magnitude of change must be understood relative to the magnitude of uncertainty in model predictions. We quantified the signal-to-noise ratio in projected distributional responses of boreal birds to climate change, and compared sources of uncertainty. Boosted regression tree models of abundance were generated for 80 boreal-breeding bird species using a comprehensive data set of standardized avian point counts (349,629 surveys at 122,202 unique locations) and 4-km climate, land use, and topographic data. For projected changes in abundance, we calculated signal-to-noise ratios and examined variance components related to choice of global climate model (GCM) and two sources of species distribution model (SDM) uncertainty: sampling error and variable selection. We also evaluated spatial, temporal, and interspecific variation in these sources of uncertainty. The mean signal-to-noise ratio across species increased over time to 2.87 by the end of the 21st century, with the signal greater than the noise for 88% of species. Across species, climate change represented the largest component (0.44) of variance in projected abundance change. Among sources of uncertainty evaluated, choice of GCM (mean variance component = 0.17) was most important for 66% of species, sampling error (mean= 0.12) for 29% of species, and variable selection (mean =0.05) for 5% of species. Increasing the number of GCMs from four to 19 had minor effects on these results. The range of projected changes and uncertainty characteristics across species differed markedly, reinforcing the individuality of species' responses to climate change and the challenges of one-size-fits-all approaches to climate change adaptation. We discuss the usefulness of different conservation approaches depending on the strength of the climate change signal relative to the noise, as well as the dominant source of prediction uncertainty.

On the connection between RNAi and heterochromatin at centromeres.

RNA interference (RNAi) is a conserved silencing mechanism whereby double-strand RNA induces specific down-regulation of homologous sequences. In the fission yeast Schizosaccharomyces pombe, centromeric heterochromatin assembly is an RNAi-dependent process. Noncoding RNAs transcribed from pericentromeric repeat sequences are processed into short interfering RNAs (siRNAs) that direct the Argonaute-containing RNA-induced transcriptional silencing (RITS) effector complex to homologous nascent transcripts. RITS is required for H3K9 methylation by the histone methyltransferase (HMT) Clr4; conversely, H3K9 methylation can attract RITS to chromatin via binding of the chromodomain protein Chp1. This codependency has hampered dissection of the order of events and mechanisms of cross talk between the RNAi and chromatin modification machineries. To tackle this problem, we have developed systems that reconstitute heterochromatin at a euchromatic locus, using either hairpin triggers or DNA-tethered chromatin-modifying complexes. These systems reveal that RNAi is sufficient to promote heterochromatin assembly in cis and that direct recruitment of the HMT Clr4 can bypass the role of RNAi in heterochromatin assembly. We have also characterized a new pathway component, Stc1, that translates the RNAi signal into chromatin marks. We discuss the implications of these findings for our understanding of the mechanism and function of RNAi-directed heterochromatin assembly at centromeres.

Assessment of chemokine receptor function on monocytes in whole blood: In vitro and ex vivo evaluations of a CCR2 antagonist.

Inhibition of monocyte and macrophage function by targeting chemokine receptors represents an attractive strategy for therapeutic intervention in inflammatory diseases. We describe an assay to assess chemokine receptor function on whole blood monocytes by measuring chemokine stimulated change in cell shape as measured by flow cytometry. The relative potential of the chemokine receptors CCR1, CCR2, CCR5, CX(3)CR1, and CXCR4 to activate monocytes in whole blood was evaluated and compared. Analysis of MCP-1 response for monocytes in blood from numerous donors revealed that the assay method had excellent intra-donor reproducibility and sensitivity. Further, the utility of this assay to determine target engagement by chemokine receptor antagonists was demonstrated using a CCR2 antagonist in rhesus monkeys. Blockade of CCR2 on whole blood monocytes was demonstrated ex vivo on blood samples collected from rhesus monkeys administered a small molecule CCR2 antagonist (MK-0812). Using a delayed-type hypersensitivity reaction to elicit monocyte recruitment to the skin of rhesus monkeys, we also evaluated the ability of MK-0812 to block monocyte migration in vivo. Blockade of CCR2 stimulation of whole blood monocytes was correlated with the inhibition of monocyte recruitment to the skin, validating the potential to use this approach in the evaluation of dose selection for chemokine receptor antagonists clinically.

Effect of lifestyle changes on whole-body protein turnover in obese adolescents.

OBJECTIVE: To investigate the effect of lifestyle changes on whole-body protein turnover (WBPT) in obese adolescents. DESIGN/METHODS: Randomized and controlled nonpharmacological intervention study of WBPT in obese adolescents using stable isotope dilution techniques. SUBJECTS AND MEASUREMENTS: We studied a total of 21 adolescents (11 boys and 10 girls, matched for their pubertal status) of which 15 were obese (age=15.8+/-0.4 y old and BMI=38.6+/-3.3 kg/m(2)) and six were lean controls (age=16.0+/-0.4 y old and BMI=21.3+/-1.2 kg/m(2)). The obese subjects were subjected to a randomized controlled lifestyle intervention program that involved moderate physical activity and diet changes for 3 months. A group of lean age-matched subjects was also studied at baseline to compare the WBPT in obese and lean adolescents. The studies were performed during a primed, continuous infusion of L-[1-(13)C]leucine. Leucine appearance rate (Leu Ra) was used as an index of whole protein breakdown and the nonoxidative portion of leucine disposal (NOLD) as an index of whole-body protein synthesis. RESULTS: The obese groups showed significantly higher body mass index (BMI), fat mass (FM), percent body fat (%BF), fat-free mass (FFM), resting energy expenditure (REE) and WBPT compared to the lean controls. The intervention program resulted in a redistribution of the parameters of body composition without apparent changes in BMI or body weight. There was a significant decrease in WBPT in the obese intervention group, but not in the obese control group. Insulin levels also decreased significantly in the obese group after intervention but not in the obese control group, whereas the glucose concentrations remained normal in all groups at baseline and also after intervention/or control. CONCLUSIONS: Results from the current study suggest: (i). abonormalities of protein metabolism occur early in the clinical course of obesity and (ii). these abnormalities are modifiable by moderate lifestyle changes in obese adolescents. The mechanism for these changes in WBPT in obese adolescents as well as their impact on specific cardiovascular risk factors and turnover of specific proteins will require further investigation.

Low exhaled nitric oxide and a polymorphism in the NOS I gene is associated with acute chest syndrome.

Abnormalities of nitric oxide metabolism have been implicated in the pathogenesis of acute chest syndrome in subjects with sickle cell anemia. It is not known whether exhaled nitric oxide levels (FE(NO)) are abnormal in children with a history of the acute chest syndrome (ACS). We compared FE(NO), plasma nitric oxide metabolites (NO(x)), serum arginine and citrulline levels, and the number of AAT repeats in intron 20 of NOS I in subjects with sickle cell disease (SCD) and a history of at least one episode of ACS (ACS(+), n = 13), subjects with SCD and no prior history of ACS (ACS(-), n = 7), and healthy children (HC, n = 6). Mean +/- SD FE(NO) (ppb) was lower in ACS(+) than in ACS(-) and HC: (10.4 +/- 4.3 versus 23.4 +/- 6.1 p = 0.002] and 30.4 +/- 15.8 [p = 0.0001], respectively). Plasma NO(x) (microM) were similar in all three groups (37.3 +/- 19.4, 33.0 +/- 13.2, 44.7 +/- 7.8, respectively). Arginine and citrulline levels (microM) did not differ between ACS(+) and ACS(-) groups. Spirometric data revealed a mildly diminished FEV(1) and FVC in ACS(+) that was statistically different from HC but not ACS(-): (FEV(1) as % of predicted for ACS(+), ACS(-), and HC; 83 +/- 17 versus 87 +/- 16 versus 102 +/- 16, respectively, p < 0.05 between ACS(+) and HC). The level of FE(NO) was significantly associated with the sum of AAT repeats in intron 20 of NOS I gene alleles. The correlation coefficient (r) was 0.62 (p < 0.005). We conclude that FE(NO) levels are significantly reduced in subjects who have a history of ACS and that the FE(NO) levels are significantly correlated with the number of NOS I AAT repeats. FE(NO) is a sensitive marker and may be a predictor of ACS prone children.

Immunolocalization of 5alpha-reductase isozymes in acne lesions and normal skin.

BACKGROUND: Dihydrotestosterone mediates androgen-dependent diseases, such as acne, hirsutism, and androgenetic alopecia. This hormone is produced from testosterone by the 5alpha-reductase enzyme. There are 2 isozymes of 5alpha-reductase (types 1 and 2) that differ in their localization within the body and even within the skin. Activity of the type 1 isozyme predominates in sebaceous glands, where it may be involved in regulation of sebum production. Since specific inhibition of 5alpha-reductase type 1 may represent a novel therapeutic approach to acne, it is important to define the localization of these isozymes in normal sebaceous follicles and acne lesions. OBSERVATIONS: Skin biopsy specimens were obtained from the backs of 11 subjects: 8 with acne and 3 without acne. Sections of normal follicles, open comedones, closed comedones, and inflammatory lesions were incubated with antibodies to types 1 and 2 5alpha-reductase. In all samples, the type 1 antibody localized specifically to sebaceous glands, and the type 2 antibody localized to the companion layer of the hair follicle (the innermost layer of the outer root sheath) and granular layer of the epidermis. Localization of the type 2 isozyme was also noted within the walls of open and closed comedones and in endothelial cells from sections of inflammatory lesions. CONCLUSIONS: The immunolocalization of 5alpha-reductase isozymes in normal sebaceous follicles and acne follicles is similar to the pattern described in terminal hair follicles and corresponds with the findings of biochemical studies that have demonstrated predominance of type 1 activity in sebaceous glands. The function of type 2 5alpha-reductase in comedones or endothelial cells in inflammatory lesions is unknown.

Immunohistochemical localization of types 1 and 2 5alpha-reductase in human scalp.

The predominant form of 5alpha-reductase (5aR) in human scalp is 5aR1. None the less, clinical studies have shown that finasteride, a selective inhibitor of 5aR2, decreases scalp dihydrotestosterone and promotes hair growth in men with androgenetic alopecia. Immunolocalization studies were thus carried out to examine 5aR isozyme distribution within scalp and, in particular, to determine whether 5aR2 might be associated with hair follicles. 5aR2 was localized using both a rabbit polyclonal and a mouse monoclonal antibody. 5aR1 was detected with a mouse monoclonal antibody. The specificity of these reagents was demonstrated both by immunofluorescence and Western blot analyses of COS cells overexpressing human 5aR1 or 5aR2. When cryosections of scalp from men with androgenetic alopecia were stained with antibody against 5aR2, using immunoperoxidase avidin-biotin complex methodology, immunostaining was observed in the inner layer of the outer root sheath and, in more proximal regions of the follicle, in the inner root sheath. Staining was also prominent in the infundibular region of the follicle, with less intense staining extending throughout the granular layer of the epidermis. Some staining was also seen in sebaceous ducts. Similar results were obtained with both the polyclonal and monoclonal 5aR2 antibodies. In contrast, in scalp cryosections stained with antibody to 5aR1, no immunostaining was observed within hair follicles. Intense staining for the type 1 isozyme was, however, detected within sebaceous glands. Our immunolocalization data suggest that the results seen in clinical trials of men with male pattern hair loss treated with finasteride may be due, at least in part, to local inhibition of 5aR2 within the hair follicle.

Active matrix metalloproteinases are present in cartilage during immune complex-mediated arthritis: a pivotal role for stromelysin-1 in cartilage destruction.

The involvement of immune complexes during experimental arthritis in induction of metalloproteinases (MMP)-induced neoepitopes in aggrecan in cartilage, as well as the role of stromelysin-1 (SLN-1) in the induction of this neoepitope, was investigated. Passive immune complex arthritis was induced, and generation of the MMP-specific cleavage product (VDIPEN) was studied by immunolocalization. The role of SLN-1 was studied with use of SLN-1-deficient (SLN-1KO) mice. VDIPEN expression was studied in vitro by exposing the cartilage to IL-1 and subsequent activation of latent MMPs. Immune complex arthritis was characterized by an acute inflammation, with influx of mainly polymorphonuclear cells into the joint cavity. Expression of VDIPEN neoepitopes was consistently found in areas extensively depleted from proteoglycans. SLN-1KO mice did not show expression of the VDIPEN neoepitope, although inflammation and proteoglycan depletion was comparable to wild-type mice. In addition, erosions of cartilage were absent in SLN-1KO mice, but were present in wild-type mice, suggesting an important role for SLN-1 in cartilage destruction. In vitro studies showed that SLN-1 is also pivotally involved in IL-1-induced MMP activity. Stimulated polymorphonuclear neutrophils were able to activate latent MMPs present in the cartilage. Neutrophil elastase was also capable of activating IL-1-induced latent MMPs, which identifies elastase as a possible activator for latent VDIPEN-inducing MMPs. This study suggests that IC are important in the activation of latent MMPs in cartilage, possibly through polymorphonuclear neutrophil activation on the cartilage edge. SLN-1 is a pivotal enzyme in overall MMP-activity in cartilage during immune complex-mediated arthritis.

Cleavage of aggrecan at the Asn341-Phe342 site coincides with the initiation of collagen damage in murine antigen-induced arthritis: a pivotal role for stromelysin 1 in matrix metalloproteinase activity.

OBJECTIVE: The destruction of articular cartilage during arthritis is due to proteolytic cleavage of the extracellular matrix components. This study investigates the kinetic involvement of metalloproteinases (MMPs) in the degradation of the 2 major cartilage components, aggrecan and type II collagen, during murine antigen-induced arthritis (AIA). In addition, the role of stromelysin 1 (SLN-1) induction of MMP-induced neoepitopes was studied. METHODS: VDIPEN neoepitopes in aggrecan and collagenase-induced COL2-3/4C neoepitopes in type II collagen were identified by immunolocalization. Stromelysin 1-deficient knockout (SLN1-KO) mice were used to study SLN-1 involvement. RESULTS: In AIA, the VDIPEN epitopes in aggrecan appeared after initial proteoglycan (PG) depletion. The collagenase-induced type II collagen neoepitopes colocalized with VDIPEN epitopes. Remarkably, cartilage from arthritic SLN1-KO mice showed neither the induction of VDIPEN nor collagen cleavage-site neoepitopes during AIA, suggesting that stromelysin is a pivotal mediator in this process. PG depletion, as measured by the loss of Safranin O staining, was similar in SLN1-KO mice and wild-type strains. Furthermore, in vitro induction of VDIPEN epitopes in aggrecan and COL2-3/4C epitopes in type II collagen, on exposure of cartilage to interleukin-1, could not be accomplished in SLN1-KO mice, whereas intense staining was achieved for both epitopes in cartilage of wild-type strains. CONCLUSION: This study emphasizes that SLN-1 is essential in the induction of MMP-specific aggrecan and collagen cleavage sites during AIA. It suggests that SLN-1 is not a dominant enzyme in PG breakdown, but that it activates procollagenases and is crucial in the initiation of collagen damage.

Kinetics of aggrecanase- and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis.

OBJECTIVE: Two major cleavage sites, one mediated by metalloproteinases (MMPs) and the other by an as-yet unidentified enzyme termed aggrecanase, have been observed in aggrecan. To learn more about the relative contribution of these enzymes during cartilage degradation, this study assessed the occurrence of both specific neoepitopes in cartilage during murine arthritis and examined the correlation between neoepitope formation and different aspects of cartilage damage. METHODS: Reversible cartilage damage was induced in mice in the zymosan-induced arthritis (ZIA) model, partly irreversible cartilage damage in the antigen-induced arthritis (AIA) model, and irreversible, destructive cartilage damage in the collagen-induced arthritis (CIA) model. Immunolocalization techniques were used to detect the specific C-terminal neoepitopes VDIPEN (MMPS) and NITEGE (aggrecanase). RESULTS: In normal cartilage from young adult mice, no VDIPEN epitopes were detected, but a limited amount of NITEGE epitopes were already present. During the early phase of proteoglycan (PG) depletion, NITEGE expression was raised substantially in all arthritis models. VDIPEN epitopes were not detected in this early phase of cartilage destruction. When PG depletion progressed toward advanced cartilage damage, VDIPEN epitopes were induced. During ZIA, minimal induction of VDIPEN was observed, whereas in AIA, strong, but partly reversible, VDIPEN staining was evident, and in CIA, an extensive presence and persistence of the MMP-induced neoepitope was seen. When VDIPEN epitopes were intensely present, NITEGE epitopes were greatly reduced at that site in the cartilage. CONCLUSION: Presence of VDIPEN epitopes in cartilage correlated with severe cartilage damage, but these epitopes were not detected during early PG degradation. This suggests a limited role for VDIPEN-inducing MMPs in early PG degradation during murine arthritis. In contrast, aggrecanase epitopes were induced before the appearance of VDIPEN epitopes, but they disappeared with progression of cartilage damage.

Increased vulnerability of postarthritic cartilage to a second arthritic insult: accelerated MMP activity in a flare up of arthritis.

OBJECTIVE: Murine antigen induced arthritis (AIA) is a chronic, smouldering inflammation. Flares of arthritis can be induced by antigen rechallenge or exposure to inflammatory mediators like interleukin 1 (IL1). These flares are characterised by a fast and marked proteoglycan (PG) depletion if compared with the initial arthritis. This study investigated the involvement of metalloproteinases in both the initial and the flare phase of arthritis. METHODS: Murine AIA was induced and a flare up of arthritis was induced by injection of 10 ng of IL1beta. Messenger RNA levels of MMP-1 and -3 were studied by RT-PCR. MMP activity in cartilage, during both primary AIA as well as the flare up of arthritis, was studied by immunodetection of MMP specific neoepitopes in aggrecan (VDIPEN). Cartilage just before flare induction was analysed for presence of MMPs at the mRNA level as well as at the protein level by zymography. RESULTS: At the onset of AIA, a fast upregulation of mRNA for stromelysin and collagenase was noted. However, no VDIPEN epitopes were detected during this early phase of arthritis. They appeared when PG depletion was severe at day 7 of arthritis and disappeared when cartilage was repaired. IL1 injection into a knee joint at week 4 of AIA caused a flare up of arthritis, coinciding with a fast and marked PG degradation. This degradation was characterised by accelerated expression of VDIPEN epitopes if compared with the expression in primary AIA. Analysis of cartilage at week 4 of AIA showed still increased mRNA levels of MMP-1 and -3. Moreover, increased levels of latent MMPs were present as well, as APMA activation induced profound VDIPEN epitope. In vitro exposure to IL1 did show increased PG breakdown but no VDIPEN expression, suggesting that factors in addition to IL1 are needed to cause the in vivo VDIPEN expression. CONCLUSIONS: The fast and marked PG depletion seen in a flare up of AIA coincides with accelarated expression of MMP induced neoepitopes compared with expression during primary AIA. This accelerated expression is probably linked to increased levels of latent enzyme, which were found to be present in the cartilage before induction of a flare up.

Interleukin-1 receptor antagonist prevents expression of the metalloproteinase-generated neoepitope VDIPEN in antigen-induced arthritis.

OBJECTIVE: To investigate the relationship between occurrence of the matrix metalloproteinase-generated neoepitope VDIPEN and proteoglycan (PG) loss in arthritis, and to examine the role of interleukin-1 (IL-1) in VDIPEN expression. METHODS: VDIPEN expression was investigated in murine antigen-induced arthritis by immunolocalization studies on joint sections. The involvement of IL-1 in VDIPEN expression was studied by blocking of IL-1 using IL-1 receptor antagonist (IL-1Ra). RESULTS: Profound PG loss was evident early in arthritis, without significant VDIPEN expression. Full expression of the neoepitope appeared after a few days, when PG depletion was severe, and disappeared at late stages when cartilage showed recovery from PG depletion. At sites where chondrocyte death occurred and cartilage did not recover from the initial cartilage depletion, VDIPEN expression remained present. Prophylactic IL-1Ra treatment of arthritic mice resulted in almost complete prevention of VDIPEN expression. However, IL-1Ra had only a minor effect on PG depletion, emphasizing that there is no correlation between VDIPEN and early PG depletion. CONCLUSION: This study indicates that IL-1 is involved in VDIPEN expression. Although VDIPEN-inducing metalloproteinases do not seem to be involved in early PG depletion during antigen-induced arthritis, metalloproteinase neoepitopes are present when PG depletion is severe.

Susceptibility of stromelysin 1-deficient mice to collagen-induced arthritis and cartilage destruction.

OBJECTIVE: It has long been proposed that stromelysin is one of the major degradative matrix metalloproteinases responsible for the loss of cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA). This hypothesis was tested by examining the arthritic paws of stromelysin 1 (SLN1)-deficient mice for loss of cartilage and for generation of neoepitopes that would be indicative of aggrecan cleavage. METHODS: The SLN1 gene was inactivated in murine embryonic stem cells, and knockout mice deficient in SLN1 activity were bred onto the B10.RIII background. The incidence and severity of collagen-induced arthritis (CIA) were compared in wild-type and knockout mice. Paws from mice with CIA were examined for loss of cartilage and for proteoglycan staining, as well as for the generation of the neoepitope FVDIPEN341. RESULTS: SLN1-deficient mice developed CIA, as did the wild-type N2 mice. Histologic analyses demonstrated no significant differences among the B10.RIII, wild-type, and knockout mice in loss of articular cartilage and proteoglycan staining. No decrease in the FVDIPEN341 epitope was observed in the SLN1-deficient mice. CONCLUSION: Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

Aggrecanase and metalloproteinase-specific aggrecan neo-epitopes are induced in the articular cartilage of mice with collagen II-induced arthritis.

OBJECTIVE: To analyze the roles of two classes of proteinases, 'aggrecanase', and matrix metalloproteinases (MMPs), in chondrodestruction during murine collagen-induced arthritis (CIA). METHODS: Generation of the 'aggrecanase' neo-epitope (NITEGE373), and the MMP neo-epitope (VDIPEN341) within aggrecan was studied by immunoperoxidase microscopy using specific anti-peptide antibodies in normal and stromelysin-1 (SLN-1) deficient knockout mice with CIA. RESULTS: High levels of NITEGE373 and VDIPEN341 neo-epitopes were observed in foci within CIA paw articular cartilage exhibiting depletion of glycosaminoglycans, in advance of significant cartilage erosion. The highest concentrations of NITEGE373 and VDIPEN341 labeling were observed and often co-distributed in the chondrocyte pericellular matrix, suggesting that stimulated chondrocytes can synthesize and/or activate both enzymes. Other regions of the cartilage frequently exhibited either NITEGE373 or VDIPEN341 labeling, but not both neo-epitopes simultaneously, suggesting that 'aggrecanase' and MMP cleavages of aggrecan may be generated independently. No detectable differences were observed in expression or distribution of either neo-epitope in SLN-1 knockout versus wild-type mice. In addition, in vitro digestion of joint sections with SLN-1 did not alter the expression of cartilage NITEGE373, while markedly increasing VDIPEN341 labeling. Peripheral nerves and brains of naive mice also exhibited intense anti-NITEGE373 labeling. CONCLUSIONS: These data indicate that NITEGE373 and VDIPEN341 aggrecan neo-epitopes are sensitive and specific markers of early joint pathology, and are consistent with the hypothesis that SLN-1 does not have 'aggrecanase' activity, and that 'aggrecanase' is distinct from the MMPs which cleave aggrecan at the MMP site.

Protein and energy metabolism in prepubertal children with sickle cell anemia.

We hypothesized that, in children with homozygous sickle cell anemia (HbSS), the shortened life-span of erythrocytes places an increased demand on protein stores, accelerates whole body protein turnover, and consequently, energy expenditure, as well as the rate of utilization of glutamine, a major fuel for reticulocytes. Eight (11.2 +/- 0.4 y old) children with HbSS who were free of infection of vaso-occlusive disease, and seven (11.3 +/- 0.4 y old) healthy black children were therefore studied in the postabsorptive state. Each received a continuous 4-h infusion of L-[1-(13)C]leucine to determine the rate of leucine oxidation, leucine rate of appearance, and nonoxidative leucine disposal, indicators of whole body protein breakdown and synthesis, respectively. Infusion of L-[2-(15)N]glutamine was used to assess rates of glutamine utilization. Resting energy expenditure and cardiac output were measured using indirect calorimetry and echocardiography, respectively. Compared with control subjects, HbSS children had a 58 and 65% higher leucine rate of appearance and nonxidative leucine disposal, respectively (both p < 0.001), 47% higher rates of whole body glutamine utilization (p < 0.01), 19% higher resting energy expenditure (p < 0.05), and 66% higher cardiac output (p < 0.01). In conclusion, children with HbSS show evidence of hypermetabolism with regard to protein, energy, and glutamine utilization. Both increased Hb synthesis and increased cardiac workload may contribute to excess protein and energy utilization. Whatever the mechanism of hypermetabolism, the data suggest that children with HbSS may have greater protein and energy requirements than the general population.

Comparison of transtracheal and extravascular Doppler determinations of stroke volume and cardiac output at various states of volume loading in piglets.

OBJECTIVE: To assess the applicability of a new technology in neonates. Transtracheal Doppler and extravascular Doppler determinations of stroke volume and cardiac output were compared with thermodilution measurements at various states of volume loading in an animal model. DESIGN: Prospective, descriptive study. SETTING: Animal research laboratory at a university medical center. SUBJECTS: Fourteen newly weaned piglets, weighing 2.8 to 6.5 kg. INTERVENTIONS: Doppler probes were placed on the endotracheal tube tip (transtracheal Doppler) and directly on the aortic adventitia (extravascular Doppler). A 4-Fr thermodilution catheter was inserted in the pulmonary artery. Stroke volume and cardiac output determinations were recorded at baseline, after a 15-mL/kg volume load and after successive 15-mL/kg blood withdrawals to exsanguination or a systolic blood pressure of < 20 mm Hg. MEASUREMENTS AND MAIN RESULTS: Transtracheal and extravascular Doppler measurements of cardiac output were not significantly different from thermodilution at any physiologic state. These techniques were able to measure stroke volumes and cardiac outputs at the low levels seen in severe hemorrhagic shock. CONCLUSIONS: Transtracheal Doppler and extravascular Doppler measurements of cardiac output compare favorably with thermodilution. These methods effectively followed trends from alterations in intravascular volume, even at very high heart rates and small stroke volumes. Transtracheal Doppler and extravascular Doppler should yield useful information in critically ill neonatal patients, where data regarding stroke volume and cardiac output may be useful in clinical management.

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

In vivo effects of stromelysin on the composition and physical properties of rabbit articular cartilage in the presence and absence of a synthetic inhibitor.

OBJECTIVE: To characterize the effects of intraarticular injection of recombinant human stromelysin (SLN) on the matrix composition and physical properties of cartilage from lapine stifle joints and the modulation of these effects by the systemic administration of an N-carboxyalkyl synthetic matrix metalloproteinase inhibitor, L-696,418. METHODS: Female 6-8-week-old New Zealand white rabbits received an intraarticular injection of 100 micrograms activated SLN in 1 stifle joint and buffer in the contralateral control knee; these animals were killed after 1 hour. A separate group of animals received an intravenous injection of either 30 mg/kg L-696,418 or buffer prior to intraarticular injection of SLN. Joints were dissected and analyzed for proteoglycan (PG) loss into joint fluid, tissue biochemical composition, and histology by toluidine blue or anti-VDIPEN antibody staining, or were frozen for physical property analysis. Disks of femoropatellar groove cartilage were harvested from the stifle joint and tested in uniaxially confined compression for determination of electromechanical and mechanical properties. RESULTS: Lapine stifle joints that received injection of SLN without systemic administration of L-696,418 showed a 13-fold increase in loss of PG into synovial fluid. Cartilage from these joints showed significant decreases in streaming potential at 1 Hz and electrokinetic coupling coefficient, but no change in equilibrium modulus, dynamic stiffness, or hydraulic permeability. Systemic treatment with L-696,418 resulted in a significant decrease in loss of PG into joint fluid and elimination of changes in cartilage high-frequency streaming potential and coupling coefficient in joints that were injected with SLN. CONCLUSION: The 1-hour exposure to SLN in vivo resulted in loss of PG and exposure of the VDIPEN epitope of the aggrecan core protein in the superficial region of the tissue near the articular surface. This highly localized degradation resulted in electromechanical behavior changes, but little or no change occurred in mechanical properties. Systemic administration of L-696,418 significantly decreased loss of PG from cartilage and prevented the highly localized tissue degradation and the resultant changes in electromechanical behavior caused by intraarticular SLN injection.

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

OBJECTIVE: To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo. METHODS: The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay. RESULTS: Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor. CONCLUSION: These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.