A PIM₂ analogue suppresses allergic airway disease

Two approaches for the synthesis of a phosphatidylinositol dimannoside (PIM₂) analogue 4 that mimics the suppressive activity of natural PIMs and also synthetic PIM₂ have been developed. This analogue, where the inositol core was replaced by glycerol, was tested for its ability to suppress cellular inflammation in a mouse model of allergic asthma and shown to be effective in suppressing airway eosinophilia. Suppression of all inflammatory cells monitored was observed, indicating a general blockade of cellular activity. These data indicate that the inositol core is not essential for this suppressive activity.     

The increasing burden of imported chronic hepatitis B--United States, 1974-2008

Background

Without intervention, up to 25% of individuals chronically infected with hepatitis B virus (HBV) die of late complications, including cirrhosis and liver cancer. The United States, which in 1991 implemented a strategy to eliminate HBV transmission through universal immunization, is a country of low prevalence. Approximately 3,000–5,000 U.S.-acquired cases of chronic hepatitis B have occurred annually since 2001. Many more chronically infected persons migrate to the United States yearly from countries of higher prevalence. Although early identification of chronic HBV infection can reduce the likelihood of transmission and late complications, immigrants are not routinely screened for HBV infection during or after immigration.

Methods

To estimate the number of imported cases of chronic hepatitis B, we multiplied country-specific prevalence estimates by the yearly number of immigrants from each country during 1974–2008.

Results

During 1974–2008, 27.9 million immigrants entered the U.S. Sixty-three percent were born in countries of intermediate or high chronic hepatitis B prevalence (range 2%–31%). On average, an estimated 53,800 chronic hepatitis B cases were imported to the U.S. yearly from 2004 through 2008. The Philippines, China, and Vietnam contributed the most imported cases (13.4%, 12.5%, and 11.0%, respectively). Imported cases increased from an estimated low of 105,750 during the period 1974–1977 to a high of 268,800 in 2004–2008.

Conclusions

Imported chronic hepatitis B cases account for approximately 95% of new U.S. cases. Earlier case identification and management of infected immigrants would strengthen the U.S. strategy to eliminate HBV transmission, and could delay disease progression and prevent some deaths among new Americans.     

GRAZING HISTORY,DEFOLIATION, AND FREQUENCY-DEPENDENT COMPETITION: EFFECTS ON TWO NORTH AMERICAN GRASSES

Agropyron smithii and Bouteloua gracilis plants from intensively grazed prairie dog colonies and from a grazing exclosure in Wind Cave National Park, South Dakota, were used to compare responses of conspecific populations with different histories of exposure to grazing and to competition for light. In separate experiments for each species, plants grown in monocultures and two-population replacement-series mixtures were used to examine effects of defoliation, frequency-dependent competition, and population on biomass and morphology. Colony and exclosure plants frequently responded differently. Defoliation more often adversely affected exclosure plants than colony plants, while interpopulation competition more often adversely affected colony plants. Defoliation frequently negated the competitive advantage of exclosure plants. Intrapopulation competition appeared to be greater among exclosure than colony plants. Our results indicate that conclusions based on studies of plants in long-term exclosures may not apply to plant populations having long histories of intensive grazing. While there were differences between species, in both, these experiments provide evidence of population differentiation, resulting in morphologically dissimilar populations which responded differently to defoliation and to inter- and intrapopulation competition.     

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography

Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.     

Femtomolar transition state analogue inhibitors of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli

Escherichia coli 5'-methylthioadenosine/S-adenosyl-homocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH). 5'-Methylthioadenosine (MTA) is a by-product of polyamine synthesis and SRH is a precursor to the biosynthesis of one or more quorum sensing autoinducer molecules. MTAN is therefore involved in quorum sensing, recycling MTA from the polyamine pathway via adenine phosphoribosyltransferase and recycling MTR to methionine. Hydrolysis of MTA by E. coli MTAN involves a highly dissociative transition state with ribooxacarbenium ion character. Iminoribitol mimics of MTA at the transition state of MTAN were synthesized and tested as inhibitors. 5'-Methylthio-Immucillin-A (MT-ImmA) is a slow-onset tight-binding inhibitor giving a dissociation constant (K(i)(*)) of 77 pm. Substitution of the methylthio group with a p-Cl-phenylthio group gives a more powerful inhibitor with a dissociation constant of 2 pm. DADMe-Immucillins are better inhibitors of E. coli MTAN, since they are more closely related to the highly dissociative nature of the transition state. MT-DADMe-Immucillin-A binds with a K(i)(*) value of 2 pm. Replacing the 5'-methyl group with other hydrophobic groups gave 17 transition state analogue inhibitors with dissociation constants from 10(-12) to 10(-14) m. The most powerful inhibitor was 5'-p-Cl-phenylthio-DADMe-Immucillin-A (pClPhT-DADMe-ImmA) with a K(i)(*) value of 47 fm (47 x 10(-15) m). These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9-91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues.     

Noble metals strip peptides from class II MHC proteins

Class II major histocompatibility complex (MHC) proteins are essential for normal immune system function but also drive many autoimmune responses. They bind peptide antigens in endosomes and present them on the cell surface for recognition by CD4(+) T cells. A small molecule could potentially block an autoimmune response by disrupting MHC-peptide interactions, but this has proven difficult because peptides bind tightly and dissociate slowly from MHC proteins. Using a high-throughput screening assay we discovered a class of noble metal complexes that strip peptides from human class II MHC proteins by an allosteric mechanism. Biochemical experiments indicate the metal-bound MHC protein adopts a 'peptide-empty' conformation that resembles the transition state of peptide loading. Furthermore, these metal inhibitors block the ability of antigen-presenting cells to activate T cells. This previously unknown allosteric mechanism may help resolve how gold(I) drugs affect the progress of rheumatoid arthritis and may provide a basis for developing a new class of anti-autoimmune drugs.     

DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation

BACKGROUND: Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS: When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.     

Estimating the impact of newly arrived foreign-born persons on tuberculosis in the United States

Background

Among approximately 163.5 million foreign-born persons admitted to the United States annually, only 500,000 immigrants and refugees are required to undergo overseas tuberculosis (TB) screening. It is unclear what extent of the unscreened nonimmigrant visitors contributes to the burden of foreign-born TB in the United States.

Methodology/Principal Findings

We defined foreign-born persons within 1 year after arrival in the United States as “newly arrived”, and utilized data from U.S. Department of Homeland Security, U.S. Centers for Disease Control and Prevention, and World Health Organization to estimate the incidence of TB among newly arrived foreign-born persons in the United States. During 2001 through 2008, 11,500 TB incident cases, including 291 multidrug-resistant TB incident cases, were estimated to occur among 20,989,738 person-years for the 1,479,542,654 newly arrived foreign-born persons in the United States. Of the 11,500 estimated TB incident cases, 41.6% (4,783) occurred among immigrants and refugees, 36.6% (4,211) among students/exchange visitors and temporary workers, 13.8% (1,589) among tourists and business travelers, and 7.3% (834) among Canadian and Mexican nonimmigrant visitors without an I-94 form (e.g., arrival-departure record). The top 3 newly arrived foreign-born populations with the largest estimated TB incident cases per 100,000 admissions were immigrants and refugees from high-incidence countries (e.g., 2008 WHO-estimated TB incidence rate of ≥100 cases/100,000 population/year; 235.8 cases/100,000 admissions, 95% confidence interval [CI], 228.3 to 243.3), students/exchange visitors and temporary workers from high-incidence countries (60.9 cases/100,000 admissions, 95% CI, 58.5 to 63.3), and immigrants and refugees from medium-incidence countries (e.g., 2008 WHO-estimated TB incidence rate of 15–99 cases/100,000 population/year; 55.2 cases/100,000 admissions, 95% CI, 51.6 to 58.8).

Conclusions/Significance

Newly arrived nonimmigrant visitors contribute substantially to the burden of foreign-born TB in the United States. To achieve the goals of TB elimination, direct investment in global TB control and strategies to target nonimmigrant visitors should be considered.     

Gender differences in UV-induced inflammation and immunosuppression in mice reveal male unresponsiveness to UVA radiation

Immunosuppression attributed mainly to the UVB (290-320 nm) waveband is a prerequisite for skin cancer development in mice and humans. The contribution of UVA (320-400 nm) is controversial, but in mice UVA irradiation has been found to antagonise immunosuppression by UVB. In other studies of photoimmune regulation, protection mediated via oestrogen receptor-β signalling was identified as a normal endogenous defence in mice, and was shown to depend on UVA irradiation. A gender bias in photoimmune responsiveness was thus suggested, and is tested in this study by comparing the UV-induced inflammatory and immune responses in male and female hairless mice. We report that male mice, which show greater skin thickness than females, developed a less intense but slower resolving sunburn inflammatory oedema, correlated with reduced epidermal expression of pro-inflammatory IL-6 than females following solar simulated UV (SSUV, 290-400 nm) exposure. On the other hand, the contact hypersensitivity reaction (CHS) was more severely suppressed by SSUV in males, correlated with increased epidermal expression of immunosuppressive IL-10. Exposure to the UVB waveband alone, or to cis-urocanic acid, suppressed CHS equally in males and females. However, whereas UVA irradiation induced immunoprotection against either UVB or cis-urocanic acid in females, this protection was significantly reduced or abrogated in males. The results indicate that males are compromised by a relative unresponsiveness to the photoimmune protective effects of UVA, alone or as a component of SSUV. This could explain the known gender bias in skin cancer development in both mice and humans.