The epidemiology of lymphangioleiomyomatosis in Japan: a nationwide cross-sectional study of presenting features and prognostic factors

BACKGROUND AND OBJECTIVE: To evaluate the characteristics and prognostic factors of Japanese patients with lymphangioleiomyomatosis (LAM). METHODS: A nationwide survey to identify patients with LAM was conducted by questionnaire. Survival probability was estimated using the Kaplan-Meier method, and the prognostic factors were analysed by Cox regression. RESULTS: Data were collected on 173 patients with pulmonary LAM. The major presenting features were pneumothorax (43%) and exertional dyspnoea (37%). The survival probabilities for patients presenting with exertional dyspnoea (Group A) were 85%, 60% and 47% after 5, 10 and 15 years, respectively, and for patients presenting with pneumothorax (Group B) were 95%, 89% and 89%, respectively. Although the age at symptom onset was higher among patients in Group A than in Group B, Cox regression revealed that the presenting feature was a prognostic factor independent of age at symptom onset (Group A/B hazard ratio = 5.732, P < 0.01). In the subgroup of patients whose initial FEV(1) was >1000 mL, or FEV(1)/FVC >40%, or %DL(CO) >40%, the rate of deterioration in these tests was greater in Group A than in Group B (P < 0.01 for FEV(1), P < 0.05 for FEV(1)/FVC and %DL(CO)). CONCLUSIONS: There are two possible subgroups of LAM patients. One subgroup that presented with pneumothorax, had onset of symptoms at a younger age and a more favourable prognosis; the other presented with exertional dyspnoea, had onset of symptoms at an older age and a poorer prognosis.     

Characterization of pathogenic T cells and autoantibodies in C-protein-induced autoimmune polymyositis

Although autoimmune processes may take place in human polymyositis, little is known with regard to its pathogenesis due to the lack of appropriate animal models. In the present study, we developed experimental autoimmune myositis (EAM) in Lewis rats by immunization with recombinant skeletal C-protein and examined the role of pathogenic T cells and autoantibodies. Using recombinant proteins and synthetic peptides, we demonstrated that skeletal C-protein Fragment 2 (SC2) has the strongest myositis-inducing ability and that myositis-inducing epitope(s) reside within the residues 334-363 of SC2 (SC2P3). However, immunization with SC2P3 induced only mild EAM compared with SC2 immunization. Characterization of T cells and antisera revealed that SC2P3 and SC2P7 contain the B cell epitope, while the T cell epitope resides in SC2P5. Furthermore, anti-SC2, but not anti-SC2P3, antisera contained antibodies against the conformational epitope(s) in the SC2 molecule. However, SC2P3 or SC2P5 immunization plus anti-SC2 antibody transfer aggravated the disease only slightly. These findings suggest that C-protein-induced EAM is formed by activation of C-protein-specific T cells along with antibodies against conformational epitopes in C-protein but that there are undetermined factors related to the disease progression. Further analysis of C-protein-induced EAM will provide useful information to elucidate the pathomechanisms of human polymyositis.     

Anti-fibrillogenic and fibril-destabilizing activities of anti-Parkinsonian agents for alpha-synuclein fibrils in vitro

The aggregation of alpha-synuclein (alphaS) in the brain has been implicated as a critical step in the development of Lewy body diseases (LBD) and multiple system atrophy (MSA). Among the antioxidant strategies proposed, increasing evidence points to the possibility of achieving neuroprotection by dopamine agonists, as well as monoamine oxidase B inhibitors. We showed previously that the anti-Parkinsonian agents dose-dependently inhibited beta-amyloid fibrils (fAbeta)(1-40) and fAbeta(1-42) formation as well as destabilized preformed fAbetas. Using fluorescence spectroscopy with thioflavin S, electron microscopy, and atomic force microscopy, we examined the effects of anti-Parkinsonian agents, selegiline, dopamine, pergolide, bromocriptine, and trihexyphenidyl on the formation of alphaS fibrils (falphaS) and on preformed falphaS. All molecules except for trihexyphenidyl, dose-dependently inhibited the formation of falphaS. Moreover, these molecules dose-dependently destabilized preformed falphaS. The overall activity of the molecules examined was in the order of: selegiline = dopamine > pergolide > bromocriptine. These agents and other compounds related structurally could be key molecules for the development of therapeutics for LBD and MSA.     

The way forward

Good public-health decisionmaking is dependent on reliable and timely statistics on births and deaths (including the medical causes of death). All high-income countries, without exception, have national civil registration systems that record these events and generate regular, frequent, and timely vital statistics. By contrast, these statistics are not available in many low-income and lower-middle-income countries, even though it is in such settings that premature mortality is most severe and the need for robust evidence to back decisionmaking most critical. Civil registration also has a range of benefits for individuals in terms of legal status, and the protection of economic, social, and human rights. However, over the past 30 years, the global health and development community has failed to provide the needed technical and financial support to countries to develop civil registration systems. There is no single blueprint for establishing and maintaining such systems and ensuring the availability of sound vital statistics. Each country faces a different set of challenges, and strategies must be tailored accordingly. There are steps that can be taken, however, and we propose an approach that couples the application of methods to generate better vital statistics in the short term with capacity-building for comprehensive civil registration systems in the long run.     

Human combinatorial libraries yield rare antibodies that broadly neutralize hepatitis C virus

One way to dissect the antibody response to an invading microorganism is to clone the antibody repertoire from immune donors and subsequently characterize the specific antibodies. Recently, methodological advances have allowed investigations of neutralizing antibodies against hepatitis C virus (HCV) in vitro. We have investigated three human mAbs, previously isolated from an individual infected with HCV of genotype 2b, that are known to cross-react in a binding assay to the envelope E2 protein of genotypes 1a and 1b. We now report that two of them have a neutralizing activity with a breadth not previously observed. Indeed, mAbs 1:7 and A8 recognized E2 from all of the six major genotypes, and they neutralized retroviral pseudoparticles [HCV pseudoparticles (HCVpp)] carrying genetically equally diverse HCV envelope glycoproteins. Importantly, these antibodies were also able to neutralize the cell culture infectious HCV clone JFH-1 in vitro, with IC(50) values of 60 ng/ml and 560 ng/ml, respectively. The conformational epitopes of these two broadly reactive antibodies were overlapping yet distinct and involved amino acid residues in the 523-535 region of E2, known to be important for the E2-CD81 interaction. The third antibody clone, representing a dominant population in the initial screen for these antibodies, was less broadly reactive and was unable to neutralize the genotype 2a infectious clone JFH-1. Our results confirm at the clonal level that broadly neutralizing human anti-HCV antibodies can be elicited and that the region amino acids 523-535 of the HCV envelope glycoprotein E2 carries neutralizing epitopes conserved across all genotypes.     

A Common Variant of Staphylococcal Cassette Chromosome mec Type IVa in Isolates from Copenhagen,Denmark, Is Not Detected by the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.Methicillin-resistant Staphylococcus aureus (MRSA) is a common nosocomial pathogen in countries all over the world. In recent years, community-associated MRSA (CA-MRSA) has become increasingly prevalent and has shown potential to cause health care-associated bloodstream infections (8, 26). Screening and isolation of MRSA-positive patients is essential to control the transmission of MRSA in hospitals (16, 24). However, conventional detection of MRSA by culture takes at least 48 h before a preliminary result is available, and as patients in many countries are only isolated when they are recognized as MRSA positive, the risk of having already transmitted MRSA is high. The real-time PCR BD GeneOhm MRSA assay (Becton Dickinson [BD] Diagnostics GeneOhm; San Diego, CA), formerly called IDI-MRSA, is one of a number of commercial kits for rapid MRSA detection directly from nasal swabs (7) and is based on primers developed by Huletsky et al. (18). The forward primers bind to the J3 region of the staphylococcal cassette chromosome mec (SCCmec), and the reverse primer binds in the orfX region that is specific for Staphylococcus aureus. At least seven SCCmec types are known (types I to VII) (3), and several subtypes, especially of type IV, have been described (21, 27).The BD GeneOhm MRSA assay has been tested in a number of studies (4, 5, 10, 11, 13-15, 22, 23, 25, 29-31). Most studies screened hospitalized patients, but only two studies described the SCCmec types of their MRSA isolates (15, 25). Therefore, it is possible that only a few predominant hospital clones with the same SCCmec types were tested. In Denmark, different CA-MRSA clones dominate and MRSA isolates mainly harbor SCCmec types IV (85%) and V (6%) (2). In-house testing with the Huletsky primers (18) revealed that they did not amplify a PCR fragment from our most-common MRSA clone, spa t024-sequence type 8 (ST8)-IVa. Based on this finding and with the knowledge of the high number of type IV subtypes known, we were interested in finding out whether the BD GeneOhm MRSA assay could detect MRSA isolates from a collection that included mainly CA-MRSA strains. We tested 349 MRSA isolates representing variants of SCCmec types I to V. Furthermore, we chose MRSA isolates of different staphylococcal protein A (spa) types to have a broad range of genetic backgrounds, testing the hypothesis that the same SCCmec type might have minor differences in different MRSA lineages and that these differences could be in the primer regions of the assay.     

Construction of nanoscale protein particle using temperature-sensitive elastin-like peptide and polyaspartic acid chain

Temperature-responsive monodisperse spheres are useful for various in vivo and in vitro applications. Size, response temperature and biocompatibility are particularly important consideration with in vivo applications. In this work, we constructed fusion proteins of low antigenic elastin-like peptide (ELP) and a polyaspartic acid chain, and studied the particles that had a favorable size and temperature of formation of particle. From DLS analysis, we confirmed that some of them formed particles with less than 100 nm in diameter around 37 °C, while the diameter of ELPs alone is larger than 1 μm in diameter. The (PGVGV)₁₆₀D₂₂, which is composed of a short aspartic acid chain and a long ELP region, had a tendency to form large particles. The temperature of formation and collapse of the protein particle were dependent on the length of the ELP and the polyaspartic acid chain, and the concentration of proteins. The direct observation with TEM indicated that the morphologies of the particles were spherical except when (PGVGV)₁₆₀D₂₂ was used. The intensities of the environment-sensitive hydrophobic fluorescence increased at 37 °C more than 1.5 times as much as at 25 °C both in free form and modified at the ELP region. These results indicated that the polarity of the environment surround the fluorescence decreased or the movement of fluorescence was limited, and thus, implied that the ELP formed a more hydrophobic or rigid region and could hold hydrophobic drugs. These results suggest that a temperature-responsive protein particle with favorable size and temperature of formation can be constructed that is suitable for any in vitro or in vivo application.     

Evaluation of changes of cell-surface thiols as a new biomarker for in vitro sensitization test

In order to find a novel biomarker for a simple assay to predict skin sensitization, we evaluated cell-surface thiols as a biomarker reflecting intracellular signaling in THP-1 cells (human monocytic cell line). First, we found that a decrease of cell-surface thiols on hapten-treated THP-1 cells was induced in parallel with phosphorylation of p38 MAPK. Next, we confirmed that 2-mercaptoethanol in the culture medium and the differentiation state of THP-1 cells did not affect the changes of cell-surface thiols by hapten. Changes of cell-surface thiols on THP-1 cells were detected after 2 h treatment with most allergens (e.g., DNCB, NiSO₄), as well as some non-allergens (e.g., Tween80, benzalkonium chloride), though other non-allergens (e.g., SDS, glycerol) had no effect. When either a significant decrease or increase of cell-surface thiols (more than 15% in each case) was used as a criterion, the results using 36 allergens and 16 non-allergens were in good accordance with those of in vivo assays. Finally, we confirmed that ATP, which is released as a consequence of cytotoxicity, did not affect the changes of cell-surface thiols. Our results suggest that changes of cell-surface thiols may be useful for an in vitro sensitization assay, which we designate as the SH test.