Lethal and sublethal effects of toxicants on bumble bee populations: a modelling approach.

Pollinator decline worldwide is well-documented; globally, chemical pesticides (especially the class of pesticides known as neonicotinoids) have been implicated in hymenopteran decline, but the mechanics and drivers of population trends and dynamics of wild bees is poorly understood. Declines and shifts in community composition of bumble bees (Bombus spp.) have been documented in North America and Europe, with a suite of lethal and sub-lethal effects of pesticides on bumble bee populations documented. We employ a mathematical model parameterized with values taken from the literature that uses differential equations to track bumble bee populations through time in order to attain a better understanding of toxicant effects on a developing colony of bumble bees. We use a delay differential equation (DDE) model, which requires fewer parameter estimations than agent-based models while affording us the ability to explicitly describe the effect of larval incubation and colony history on population outcomes. We explore how both lethal and sublethal effects such as reduced foraging ability may combine to affect population outcomes, and discuss the implications for the protection and conservation of ecosystem services.

Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis.

Melioidosis is a severe infection caused by the flagellated bacterium Burkholderia pseudomallei. The nonsense polymorphism TLR51174C>T is associated with improved outcome in Thais with melioidosis. We hypothesized that other TLR5 variants may modulate the host response and determine outcome in melioidosis. We genotyped 12 TLR5 variants selected de novo from the HapMap database and examined the association of each with cytokines induced by flagellin stimulation of whole blood from healthy Thai subjects. We found a blunted cytokine response for three related markers that were in linkage disequilibrium (LD) with a non-synonymous variant, TLR51846T>C. Carriers of TLR51846T>C had broadly impaired cytokine responses induced by flagellin. TLR51846T>C was associated with protection against death in melioidosis patients (odds ratio: 0.62, 95% confidence interval: 0.42-0.93, P=0.021). We observed no impairment in TLR51846C-dependent nuclear factor κB activation, however, suggesting an alternative mechanism for the effect. We found that TLR51846T>C was in strong LD with TLR51174C>T. Many of the blunted cytokine responses observed and the association of TLR51846T>C with survival in melioidosis patients may be attributable to TLR51174C>T, but we could not exclude an independent effect of TLR51846T>C. These data identify novel associations for TLR51846T>C, enhance our understanding of TLR5 genetic architecture in Thais and highlight the role of TLR5 in melioidosis.

Model for the in vivo assembly of nascent Ld class I molecules and for the expression of unfolded Ld molecules at the cell surface.

To characterize the process of class I assembly and maturation, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of intracellular and surface Ld molecules can be detected in an alternative conformation designated Ldalt1. Nascent Ldalt molecules are non-peptide ligand associated and are weakly associated with beta 2-microglobulin (beta 2m). Unexpectedly, when monoclonal antibodies were added directly to the lysis buffer, significant amounts of Ldalt/beta 2m heterodimer were detected, suggesting that beta 2m association is not necessarily sufficient to induce Ld conformation. By contrast, addition of peptide to cell lysates rapidly induced the folding of beta 2m-associated Ldalt to conformed Ld. Furthermore, the time course and dynamics of this conversion correlated precisely with peptide binding to Ld. The precursor-product relationship of Ldalt and conformed Ld was also visualized in vivo by pulse-chase analysis of BALB/c splenocytes. To investigate the factors that regulate intracellular transport of class I molecules, expression of Ld was studied in the peptide transport-deficient cell line, RMA.S-Ld, and in beta 2m-/- splenocytes. In contrast to wild-type cell lines, both Ldalt and conformed Ld are poorly expressed at the cell surface of RMA.S-Ld and beta 2m-/- splenocytes. Therefore, surface expression of Ldalt is dependent upon the concomitant expression of conformed Ld molecules. To determine whether surface Ldalt molecules can result from melting of conformed Ld molecules, surface Ld molecules were loaded with several different known Ld peptide ligands. Complexes of Ld with different ligands were found to have dramatically disparate surface half-lives. Importantly, the Ld peptide complexes that turned over the most rapidly resulted in the most gain in surface Ldalt, implying that peptide dissociation can induce the accumulation of nonconformed Ld heavy chains at the cell surface.

Molecular evidence that the H-2D and H-2L genes arose by duplication. Differences between the evolution of the class I genes in mice and humans.

To resolve issues regarding the evolution of D region class I MHC genes and their relationship to other class I-encoding regions of the mouse, as well as man, we characterized the class I genes from the Dq region of the B10.AKM mouse strain. The Dq region was selected because it was known to express multiple gene products, yet two of the products previously characterized have structural features in common with the Ld molecule. Since DNA hybridization data defined similarities between the Dd and Dq regions, we used low-copy genomic or oligonucleotide probes derived from the Dd region of BALB/c (H-2d) to screen a B10.AKM cosmid library. Cosmid clones containing Dq, D2q, D3q, D4q, Lq, and Q1q genes have been isolated and aligned with the corresponding genes of the BALB/c MHC, thus demonstrating a similar gene organization. The two classical transplantation genes, Dq and Lq were found to be strikingly similar to each other such that exons 1-3 of Dq and Lq, are approximately 97% homologous, and exons 4-8 are identical. Furthermore, the implied amino acid sequences of both Lq and Dq molecules show considerable homology to Ld, particularly in regions presumed to be involved in ligand binding. These comparisons suggest not only that the Dq and Lq genes arose from the duplication of an Ld-like progenitor, but also that there is a selective advantage for the maintenance of an Ld-like structure. In addition, the 5' portion of the D4q gene was sequenced and found to have a 13-bp deletion and a 4-bp insertion within the alpha 2 exon. These result in a frame shift that creates a premature termination codon and potential polyadenylation site, respectively. Thus, D4q does not encode a typical class I molecule. Sequence comparisons suggest that the D4q gene did not arise from a duplication event involving an Ld-like gene such as Dq and Lq. Interestingly, the D4q molecule, if produced, would have amino acid residues in common with K and/or Q molecules that differ from those observed in D/L molecules. These findings, in conjunction with hybridization data, provide evidence that the D2, D3, and D4 genes were derived from Q genes by an unequal crossover event. Additional hybridization data using low-copy D region probes suggest that several different D region gene organizations exist among mice of different haplotypes. These and other recent molecular studies provide multiple examples of expansion and contraction of the class I genes in the D region.(ABSTRACT TRUNCATED AT 400 WORDS)

The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure.

To better understand the biological implications of the association of ligand with major histocompatibility complex class I molecules, we have studied the Ld molecule of the mouse. The culturing of various nonselected cell lines with three different known Ld peptide ligands resulted in a two- to fourfold specific increase in surface Ld expression as detected by 10 of 11 different monoclonal antibodies (mAbs) recognizing Ld epitopes. These findings suggest that Ld molecules are not saturated with endogenous peptide ligands and thus have accessible binding sites. Exploiting this feature of Ld we demonstrate that the physical association of Ld with ligand is exquisitely specific, indicating that they function in determinant selection. In addition, a non-peptide-bound antigenic variant of Ld was specifically detected with an exceptional mAb designated 64-3-7. In comparison with other Ld molecules, 64-3-7+ Ld molecules are not peptide ligand inducible, are more susceptible to proteolysis, lack beta 2 microglobulin association, and display a slower rate of oligosaccharide maturation. In spite of their deficiencies, the non-ligand-associated 64-3-7 Ld molecules were detected on the surface of all cell types tested; however, they appear not to be recognized by alloreactive cytotoxic T lymphocytes.

Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface.

To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non-ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding.

An expanded approach to the problem of disappearing species.

Assistance for disappearing species is at present too localized and dispersed to make much impact on the problem with its growing dimensions. Species are threatened primarily because of their status as common property. Institutional deficiencies, notably those of free markets and property rights, promote depletion of species. Conversely, present institutional mechanisms offer little scope for society to express its preferences for goods without price or to establish responsibility for common-heritage resources. The situation postulates corrective measures on the part of collective authority at the international level. These measures would require a joint commitment by the developed and developing worlds, on a scale to reflect the increasingly interdependent needs and opportunities of the community at large. Whether the community perceives itself as a community or not, it functions as such in many of its ecological relationships and economic interactions. The community will sooner or later be obliged to respond to the problem of vanishing species: either sooner, through protective measures of sufficient scope, or later, when it finds that the disappearance of large numbers of species represents a loss through which it is indivisibly impoverished.

Environmental unknowns.

Among the environmental problems ahead, the most important ones could be those that are still unknown to us. This conceptual article explores this prospect on the grounds that it is important not only to supply answers to recognized questions but to raise appropriate new questions.

National Parks in Savannah Africa: Ecological requirements of parks must be balanced against socioeconomic constraints in their environs.

A national park is as integral to its regional environment as it is to the nation. Whether one wants to manage it that way or not, a park is dependent on the resources-human and physiobiological-of the environs, just as the environs are modified by the park's existence. This view may not jibe with the spirit of those who strive to protect a patch of old-time Africa as a refuge of serenity and stability in a world of tumult and change. But the park has its own ecology, just as does any creature within it, although the park's, being more abstract, is more difficult to discern. Planned or not, a park's future is even more enmeshed with the region's future than with the nation's. The ramifications of this relationship, especially the socioeconomic ones, are not always recognized, with the result that the enmeshing process sometimes sounds like a crunching of the gears. The worlds on both sides of the park boundary would get along better if there were a clear indication of what each can do for the other. By contrast, if they spend their energy resisting one another, there is little doubt as to which must be the ultimate "winner."

Induction of M2 Macrophages Prevents Bone Loss in Murine Periodontitis Models.

Periodontitis is characterized by the progressive destruction of tooth-supporting alveolar bone, which is mainly caused by chronic inflammation in response to persistent bacterial insult. It has recently become clear that the pathogenesis of periodontitis is associated with a high ratio of proinflammatory M1 (classically activated) macrophages to anti-inflammatory M2 (alternatively activated). To decrease the inflammatory activity, we locally delivered the C-C motif chemokine ligand 2 (CCL2) using controlled-release microparticles (MPs). CCL2 is known to promote chemotaxis of M0 or M2 phenotype macrophages to the inflamed site and induce M2 phenotype polarization locally. Our in vitro data showed that CCL2 increased the number of M2 phenotype macrophages, decreased TNF-α secretion, and enhanced chemotaxis of RAW264.7 cells toward CCL2 MPs. Moreover, we induced periodontal disease in 2 animal models through inoculation of Porphyromonas gingivalis and ligature around the murine molar. Micro-computed tomography analysis showed significant reduction of alveolar bone loss in the CCL2 MP treatment group when compared with a blank MP group and a no-treatment periodontitis group in both models. Immunohistologic analysis showed a significant increase in the M2 phenotype subset and a decrease in the M1 phenotype subset in the CCL2 MP group of the P. gingivalis-induced model. Also, in both models, tartrate-resistant acidic phosphatase staining showed significantly fewer numbers of osteoclasts in the CCL2 MP group in alveolar bone area. Moreover, quantitative polymerase chain reaction results showed a significant increase in IL-1RA (interleukin 1 receptor antagonist) mRNA expression and a decrease in RANKL (receptor activator of nuclear factor kappa-Β ligand) mRNA expression in the CCL2 MP group in the ligature model. In summary, manipulation of endogenous M2 phenotype macrophages with CCL2 MPs decreased the M1 phenotype:M2 phenotype ratio and prevented alveolar bone loss in mouse periodontitis models. The delivery of CCL2 MPs provides a novel approach to treat periodontal disease.

Comparison of early postoperative rehabilitation outcome following total knee arthroplasty using different surgical approaches and instrumentation.

PURPOSE: To assess early postoperative rehabilitation outcome following computer-assisted total knee arthroplasty (TKA) or standard instrumentation TKA using a medial parapatellar or subvastus approach. METHODS: A prospective controlled trial of 70 consecutive patients undergoing TKA with a low contact stress rotating platform prosthesis was conducted. Patients were randomised to receive surgery with either computer navigation or standard instrumentation. A medial parapatellar or subvastus approach was used according to the surgeons' preference. Outcome measures included preoperative knee function, intra-operative factors, and postoperative rehabilitation. RESULTS: Duration of surgery was significantly longer when using computer navigation; however, operating time decreased with greater experience. A higher incidence and duration of early postoperative quadriceps dysfunction was associated with computer-assisted TKA through the medial parapatellar approach than through the subvastus approach or TKA performed with standard instrumentation. No patient who received surgery through the subvastus approach had a lag of more than 20 degrees, at 48 hours postoperatively, regardless of the instrumentation used. CONCLUSION: Computer-assisted TKA through a medial parapatellar approach was associated with delayed recovery of the quadriceps during early postoperative rehabilitation. This was due to the additional quadriceps dissection required to place the femoral tracking array. The subvastus approach is therefore recommended for computer-assisted TKA.

Hemodynamic findings in 123 patients with acute myocardial infarction on admission.

Hemodynamic and clinical evaluations of 123 patients with acute myocardial infarction were performed during the first hour of admission to the hospital. In the 123 patients, the right atrial pressure was less than 10 mm Hg in 49 patients, the right atrial oxygen saturation was less than 70% in 97 patients, the arteriovenous oxygen difference was greater than 5.0 vol% in 78 patients. The arterial Po2 was less than 90 mm Hg in 101 of 107 patients who could be evaluated while breathing room air. The cardiac index was depressed below 3.0 L/min/m2 in 65 of 98 patients. The hemodynamic findings generally correlated with the clinical status of the patient; however, within each clinical class of patients there was a wide spectrum of values for each measurement evaluated. There was also considerable overlap of the values found within each clinical classification. It is concluded that hemodynamic evaluation of patients with acute myocardial infarction presents a profile of the patient which is frequently different from the profile that clinical evaluation presents. An objective hemodynamic classification of patients with acute myocardial infarction may provide a more useful index for the evaluation of the patient's prognosis and for the assessment of preventative therapy.

Peptide ligand structure influences the exchange of beta2-microglobulin by cell surface Kb.

We have studied the interactions which occur between the peptide ligand and beta2-microglobulin (beta2m) components of the class I MHC complex by analysing the process of beta2m exchange. We have previously shown that the rate of beta2m exchange on a cell-surface class I MHC complex varies with the peptide ligand to which it is bound. It remains unclear, however, whether the ability of peptide ligand to alter beta2m/heavy-chain association is related to peptide affinity, peptide structure, or both. In this article, we examine the effects of variations in peptide ligand structure on the rate of beta2m exchange by cell surface Kb complexes. Using a panel of alanine substituted variants of the MCMV peptide (YPHFMPTNL), we show that single amino acid changes in peptide sequence can have dramatic effects on the rates of beta2m exchange. The observed changes in beta2m exchange rates are directly due to modification of the peptide ligand structure as they do not reflect changes in peptide affinity. These findings suggest that peptide ligand structure can induce conformational changes in the Kb heavy chain which alter the rates of cell surface beta2m exchange, and provide further evidence for peptide-dependent fluidity of the class I heavy chain.

Structural analyses define an additional H-2D region class I antigen that is expressed by a variant mouse strain.

The genetic complexity of the H-2D region includes haplotype disparities in apparent gene and product number. To probe the genetic basis of this complexity, the products of two independently derived mouse strains (STU and B10.SAA48) that express Dw3 antigens were compared. Serologic, fluorometric and peptide map comparisons were made using monoclonal antibodies. Although both STU and B10.SAA48 mice were found to express indistinguishable Dw3 molecules, only B10.SAA48 mice were found to express an additional antigen designated Lw3. Several lines of evidence are presented that suggest the gene encoding Lw3 maps to the D region. Furthermore peptide map comparisons of Dw3 with Lw3 molecules implied that they are products of separate genes; but Dw3 and Lw3 molecules were found to be more homologous to each other than Dd and Ld molecules are to each other. Inter-haplotype comparisons of Dw3 and Lw3 molecules with other D region molecules showed no striking homologies to Dd, Ld or eight other molecules compared. However, both Dw3 and Lw3 molecules were found to be unexpectedly homologous to the Ddx and Dw25 molecules, thus defining another family of structurally related D region antigens. This so called Dw3-family was found to be quite distinct from the previously defined Ld-family of molecules, since no joint members were found. The results of these studies of Dw3 encoded antigens are discussed as evidence for intra-D region recombination or mutation.

Biochemical and functional characterization of soluble multivalent MHC L(d)/Fc gamma 1 and L(d)/Fc mu chimeric proteins loaded with specific peptides.

Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)-peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen-specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H-2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes mu or gamma1. Stable transfectants of these L(d)/Fc gamma1 and L(d)/Fc mu genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/ Fc gamma1 and L(d)/Fc mu monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fc gamma1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fc gamma1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.

Esophageal disorders associated with emesis in infants and children.

Regurgitation of esophageal contents is referred to as esophageal emesis or esophageal vomiting. It is most likely to be confused with symptoms arising from disease proximal or distal to the esophagus, particularly the latter. Therefore, it is important to distinguish between esophageal vomiting and typical vomiting, although in the former group, the regurgitated contents are ingested food and/or saliva; recognition of the exact site of the pathological disorder may necessitate investigation. A wide variety of investigations may be necessary. Disease processes to be considered in the diagnosis include congenital stricture, achalasia and other motility disorders of the esophagus, foreign-body impaction, and acquired strictures of the esophagus, such as peptic or corrosive strictures.

Nutritional and metabolic characterization of a thiamine-deficient rat model.

The effects of a thiamine-deficient diet on plasma and tissue vitamin concentrations and on whole-body glucose metabolism were assessed. Male Sprague-Dawley rats (175 to 200 g body weight) fed a thiamine-deficient (TD) or nutritionally complete purified diet were used for plasma thiamine mononitrate and monophosphate and for red blood cell and tissue thiamine pyrophosphate (TPP) determinations weekly for up to 5 weeks. Additional rats were used for assessment of basal glucose kinetics by using a primed constant infusion of [3-3H]glucose. Plasma thiamine mononitrate levels decreased 60% at 1 week and were undetectable after 5 weeks on the diet. Plasma thiamine monophosphate decreased 80% after 1 week on the TD diet, and levels were undetectable after 4 weeks on the diet. Red blood cell TPP in the TD group decreased progressively with time: 54% at 1 week, 86% at 3 weeks, and 92% at 5 weeks. At 1 and 4 weeks, the decrease in tissue TPP was significant in the liver (65% and 89%, respectively), gut (52% and 94%, respectively), spleen (40% and 60%, respectively), and skeletal muscle (37% and 76%, respectively), with the brain (7% and 84%, respectively) showing the slowest initial rate of depletion. The TD diet did not alter plasma glucose concentrations, but it increased plasma lactate by 75% and plasma pyruvate by 50% to 75%. Rates of hepatic glucose production and peripheral glucose utilization were not different between the control and TD rats at 2 weeks, but they were 25% higher in the TD rats after 6 weeks on the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Portal hypertension in children--the changing spectrum.

This report is an analysis of changing trends in the etiology and management of portal hypertension (PHT) in childhood. The study compared the 53 admissions to the Royal Children's Hospital from 1971 to 1991 (33 intrahepatic, 20 extrahepatic) with the 77 admissions (22 intrahepatic, 54 extrahepatic, 1 with a congenital anomaly of the mesenteric vein) from the previous 23-year period (1948 to 1971). In addition to the differences in etiology, there has been (1) an increasing role for sclerotherapy as a therapeutic modality (and with this, lessening of the role of surgery), (2) a different approach to investigation (particularly imaging techniques), and (3) the availability of organ transplantation.