Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin

Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.     

Intestinal pseudo-obstruction as an initial presentation of systemic sclerosis in two children

Two children are reported in whom intestinal pseudo-obstruction was the initial manifestation of systemic sclerosis. Gastrointestinal symptoms and skin changes resolved or improved in both children following treatment with prednisone and penicillamine (case 1) or methotrexate (case 2), although radiological changes of the gastrointestinal tract persisted at 3 and 2 yr of follow-up, respectively.      

Preclinical to Clinical Translation of CNS Transporter Occupancy of TD-9855, a Novel Norepinephrine and Serotonin Reuptake Inhibitor

Background:

Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters.

Methods:

We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor.

Results:

TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC₅₀ of 11.7ng/mL and 50.8ng/mL, respectively, consistent with modest selectivity for NET in vivo.Accounting for species differences in protein binding, the projected human NET and SERT plasma EC₅₀ values were 5.5ng/mL and 23.9ng/mL, respectively. A single-dose, open-label PET study (4–20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [¹¹C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [¹¹C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30–40h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC₅₀ for NET was estimated to be 1.21ng/mL, and at doses of greater than 4mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35ng/mL.

Conclusions:

These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation.     

Long-term mortality and morbidity of transfusion-associated non-A, non-B, and type C hepatitis: A National Heart, Lung, and Blood Institute collaborative study

Persons with non-A, non-B hepatitis (cases) identified in 5 transfusion studies in the early 1970s have been followed ever since and compared for outcome with matched, transfused, non-hepatitis controls from the same studies. Previously, we reported no difference in all-cause mortality but slightly increased liver-related mortality between these cohorts after 18 years follow-up. We now present mortality and morbidity data after approximately 25 years of follow-up, restricted to the 3 studies with archived original sera. All-cause mortality was 67% among 222 hepatitis C-related cases and 65% among 377 controls (P = NS). Liver-related mortality was 4.1% and 1.3%, respectively (P =.05). Of 129 living persons with previously diagnosed transfusion-associated hepatitis (TAH), 90 (70%) had proven TAH-C, and 39 (30%), non-A-G hepatitis. Follow-up of the 90 TAH-C cases revealed viremia with chronic hepatitis in 38%, viremia without chronic hepatitis in 39%, anti-HCV without viremia in 17%, and no residual HCV markers in 7%. Thirty-five percent of 20 TAH-C patients biopsied for biochemically defined chronic hepatitis displayed cirrhosis, representing 17% of all those originally HCV-infected. Clinically evident liver disease was observed in 86% with cirrhosis but in only 23% with chronic hepatitis alone. Thirty percent of non-A, non-B hepatitis cases were unrelated to hepatitis viruses A,B,C, and G, suggesting another unidentified agent. In conclusion, all-cause mortality approximately 25 years after acute TAH-C is high but is no different between cases and controls. Liver-related mortality attributable to chronic hepatitis C, though low (<3%), is significantly higher among the cases. Among living patients originally HCV-infected, 23% have spontaneously lost HCV RNA.     

Trait-mediated assembly processes predict successional changes in community diversity of tropical forests

Interspecific differences in relative fitness can cause local dominance by a single species. However, stabilizing interspecific niche differences can promote local diversity. Understanding these mechanisms requires that we simultaneously quantify their effects on demography and link these effects to community dynamics. Successional forests are ideal systems for testing assembly theory because they exhibit rapid community assembly. Here, we leverage functional trait and long-term demographic data to build spatially explicit models of successional community dynamics of lowland rainforests in Costa Rica. First, we ask what the effects and relative importance of four trait-mediated community assembly processes are on tree survival, a major component of fitness. We model trait correlations with relative fitness differences that are both density-independent and -dependent in addition to trait correlations with stabilizing niche differences. Second, we ask how the relative importance of these trait-mediated processes relates to successional changes in functional diversity. Tree dynamics were more strongly influenced by trait-related interspecific variation in average survival than trait-related responses to neighbors, with wood specific gravity (WSG) positively correlated with greater survival. Our findings also suggest that competition was mediated by stabilizing niche differences associated with specific leaf area (SLA) and leaf dry matter content (LDMC). These drivers of individual-level survival were reflected in successional shifts to higher SLA and LDMC diversity but lower WSG diversity. Our study makes significant advances to identifying the links between individual tree performance, species functional traits, and mechanisms of tropical forest succession.Quantifying the relative importance of mechanisms that drive community assembly remains a central challenge in ecology. Interspecific variation in ecological strategies is considered a major driver of community assembly and has been classified by Chesson (1) into relative fitness (i.e., per capita population growth rate) differences and stabilizing niche differences (2). These processes act concurrently, and their importance may vary over space and time (3). However, the simultaneous effects of different assembly mechanisms on community dynamics have not been well-characterized, particularly in diverse communities such as tropical forests. High dynamism of vegetation composition during tropical forest succession creates an ideal opportunity to investigate the drivers of community assembly.Interspecific niche and fitness differences can be characterized through the lens of interspecific functional trait variation, an approach that can shed light on drivers of community functional composition and dynamics (2, 4, 5). Traits may mediate niche and fitness differences in at least four ways (Fig. 1). First, interspecific trait variation can be correlated with fitness independent of neighbor density, such that species possessing traits associated with the highest fitness should exclude species with unfavorable traits (1, 2) (1 in Fig. 1). Second, traits may be associated with a species’ sensitivity to neighbor density (i.e., a trait × crowding interaction effect on fitness). As a result, certain trait values may have relatively low fitness under high crowding (6–9) (2 in Fig. 1). For example, species with low wood specific gravity (WSG) might have higher fitness at the initiation of succession but be disadvantaged later in succession because of sensitivity to crowding. Third, negative density effects of neighbors may be asymmetric between species and dependent on hierarchies (i.e., species with favorable traits experience weaker negative density effects of neighbors with unfavorable traits, generating relative fitness differences) (10) (3 in Fig. 1). For example, species with high WSG may have stronger negative density effects on neighbors with low WSG than vice versa (10). Fourth, trait variation may be related to local niche differences between neighbors, such that greater trait differences weaken negative density effects of neighbors and promote stable coexistence of functionally diverse neighbors (11, 12) (4 in Fig. 1). Overall, traits associated with fitness differences (1–3 in Fig. 1) are expected to decrease in diversity over time, whereas traits associated with stabilizing niche differences (4 in Fig. 1) are expected to increase in diversity, assuming no trait bias in immigration. Gaining a more nuanced, complete, and predictive understanding of community assembly requires that we quantify the effects of these assembly processes on species demography and the associated changes at the community level (13, 14).Open in a separate windowFig. 1.Illustration of the four mechanisms of trait-mediated assembly included in our models. Assembly may be driven by species differences in (1) survival independent of crowding by neighbors, (2) response to increased crowding, (3) competitive hierarchies, and (4) local niche. All four processes may be revealed through the lens of functional trait variation (the spectrum from yellow to green) and its effects on mortality (black Xs). Note that the color schemes are not meant to be mutually compatible. In 1, the green color is associated with greater fitness (e.g., because of higher survival) compared with yellow, independent of neighborhood crowding. (2) Green traits have lower sensitivity to crowding relative to yellow traits (irrespective of neighbors'' traits, which are shown in gray). (3) Species have trait hierarchies. Here, green is dominant (i.e., greener species always have greater negative effect on the performance of yellower neighbors than vice versa). (a) Because their traits are very different, the green species has a stronger competitive effect on the yellow species compared with (b) the competitive effect of the yellow-green species on the yellow species. (4) In contrast, when trait variation is associated with stabilizing niche differences between neighbors, negative density effects are experienced similarly by both species. (a) Neighbors with greater trait-associated niche difference (green vs. yellow) have a weaker competitive effect relative to (b) species with less difference in traits (green vs. yellow-green).Successional tropical forests exhibit rapid community assembly and thus, provide an ideal system for testing assembly theory (15). High species richness and elevated tree growth and mortality rates relative to old-growth forests contribute to rapid species turnover and increase the likelihood of detecting assembly processes across succession (16). As succession proceeds, increased crowding alters local resource availability, which may drive species turnover (17–19). Early successional forests are typically dominated by species with acquisitive traits, such as low WSG and high specific leaf area (SLA), which enable rapid growth, resource capture, and high fitness under conditions of high resource availability (17, 20, 21). In contrast, traits that tend to dominate older forests [e.g., dense wood and tough leaves (22, 23)] are thought to promote long-term survival and fitness under high crowding and low resource availability (17, 24). Trait diversity is also expected to change as succession proceeds, although the direction and mechanisms of diversity changes remain unclear (23). Despite considerable previous efforts, few researchers have tested these hypotheses using demographic data in diverse systems.We use a long-term dataset of tropical forest succession to disentangle how trait-mediated processes drive community dynamics. Spatially explicit longitudinal studies of individual performance offer a powerful approach to reveal drivers of community dynamics (9, 12, 14, 25–27). Empirical studies of functional community assembly have typically compared static community patterns across sites using aggregated trait metrics (28–30). These studies assume that community patterns are the result of prior assembly processes. However, static and aggregated patterns provide limited capability to distinguish between multiple, often opposing assembly processes (14, 31).Our novel approach simultaneously quantifies the relative importance of multiple assembly processes and links these processes to contrasting effects on functional diversity. We use trait data for over 200 tree species, and we develop spatially explicit statistical models of tree survival over 15 y across eight 1-ha forest plots at diverse stages of succession in lowland wet forests of Costa Rica (SI Appendix, Table S1). We ask three central questions about trait-mediated effects on tree survival rates, a major component of fitness, and successional community dynamics.

• i)What are the simultaneous effects and relative importance of four trait-mediated assembly processes on tree survival? Specifically, we ask four subquestions about the processes.
  • i.i) How are functional traits related to interspecific variation in average survival?
  • i.ii) How do trait relationships with survival change with variation in neighborhood crowding?
  • i.iii) Do competitive interactions depend on trait hierarchy, indicating that crowding effects are based on dominance?
  • i.iv) Do competitive interactions with neighbors depend on absolute trait differences between neighbors, suggesting stabilizing niche differences?
• ii)Is the demographic evidence for trait-mediated fitness and niche differences (i) reflected in community-level changes in trait diversity across successional stages? We expect that traits associated with relative fitness (i.e., associated with greater average survival, decreased sensitivity to crowding, and hierarchical dominance) will decrease in diversity over time, whereas traits associated with stabilizing niche differences will increase in diversity during forest succession.
• iii)Is the demographic evidence for trait-mediated fitness differences reflected in functional differences between second-growth specialists and old-growth specialists? We expect that traits associated with greater relative fitness will be more strongly associated with old-growth specialists compared with second-growth specialists.

To answer these questions, we built hierarchical Bayes models of community-wide stem survival, which include parameters for each assembly process in questions i.i–i.iv (12, 27) (details in Methods). We fit models for each of three functional traits associated with resource use and life history: SLA (defined as leaf area per unit dry mass), leaf dry matter content (LDMC; defined as leaf dry mass over leaf fresh mass), and WSG (defined as the density of wood relative to the density of water). These traits represent leading axes of ecological variation among tropical tree species that have been previously implicated in interspecific variation in resource use efficiency, species interactions, and life history strategies (32–34). SLA represents a major axis of variation between rapid resource acquisition for species with high SLA vs. conservative strategies with low tissue turnover for species with low SLA (32, 35). Species with high LDMC have lower leaf protein and reduced ability to exploit resource-rich environments but better performance under low resources and drought (7, 34, 36, 37). WSG is associated with a tradeoff between rapid growth rates for species with low WSG and high structural support and resistance to natural enemies for species with high WSG (9, 33).     

Hematopoietic defects in mice lacking the sialomucin CD34

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34- null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34- null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.     

The cytoskeleton in Chediak-Higashi syndrome fibroblasts

The Chediak-Higashi syndrome (CHS) trait is expressed in cultured human skin fibroblasts as an abnormal perinuclear concentration of moderately enlarged lysosomes. The cytoskeleton of CHS fibroblasts appears intact. Microtubules are normal in number and morphology, as assessed by colchicine binding studies, antitubulin immunofluorescence, and electron microscopy. Deformability by shear force is unaltered and microfilaments are abundant. However, CHS lysosomes appear to interact abnormally with the cytoskeleton, since the perinculear aggregation partially disperses after depolymerization of cell microtubules with colchicine. These results suggest that CHS is associated with a defect of either the lysosomal membrane itself or of lysosomal membrane- microtubule interaction.     

Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.     

CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia are caused by mutations of the WAS protein (WASP) gene. WASP may be involved in the regulation of podosome, an actin-rich dynamic cell adhesion structure formed by various types of cells. The molecular links between WASP and podosomes or other cell adhesion structures are unknown. Platelets express an SH2-SH3 adapter molecule, CrkL, that can directly associate with paxillin, which is localized in podosomes. The hypothesis that CrkL binds to WASP was, therefore, tested. Results from coprecipitation experiments using anti-CrkL and GST-fusion proteins suggest that CrkL binds to WASP through its SH3 domain and that the binding was not affected by WASP tyrosine phosphorylation. The binding of GST-fusion SH3 domain of PSTPIP1 in vitro was also not affected by WASP tyrosine phosphorylation, suggesting that the binding of the SH3 domains to WASP is not inhibited by tyrosine phosphorylation of WASP. Anti-CrkL also coprecipitates a 72-kd protein, which was identified as syk tyrosine kinase, critical for collagen induced-platelet activation. CrkL immunoprecipitates contain kinase-active syk, as evidenced by an in vitro kinase assay. Coprecipitation experiments using GST-fusion CrkL proteins suggest that both SH2 and SH3 domains of CrkL are involved in the binding of CrkL to syk. WASP, CrkL, syk, and paxillin-like Hic-5 incorporated to platelet cytoskeleton after platelet aggregation. Thus, CrkL is a novel molecular adapter for WASP and syk and may potentially transfer these molecules to the cytoskeleton through association with cytoskeletal proteins such as Hic-5.