Fas (CD95/Apo-1) ligand regulation in T cell homeostasis, cell-mediated cytotoxicity and immune pathology

Members of the tumor necrosis factor (TNF) superfamily are crucially involved in the regulation of T cell activation, homeostasis and cytotoxicity. In particular, Fas ligand (FasL), expressed by activated T lymphocytes, induces cell-mediated cytotoxicity and may also be responsible for apoptotic suicide. Tight regulation of this death-inducing ligand is a prerequisite for proper immune defense and homeostasis. In this review, we will discuss various aspects of FasL regulation in cell-mediated cytotoxicity, immune homeostasis and the immunopathology of diseases.     

Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Escherichia coli

OBJECTIVES: 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing resistance-nodulation-cell division type efflux pumps, but there is no data on its activity in clinical isolates of E. coli. METHODS: The antimicrobial susceptibility of 60 clinical isolates of E. coli to a variety of antimicrobial agents was determined in the absence and presence of NMP and, for comparison, of Phe-Arg-beta-naphthylamide (PAbetaN), another putative efflux pump inhibitor (EPI). The intracellular accumulation of ethidium bromide was measured to confirm efflux pump inhibition as the likely mechanism of action of NMP. RESULTS: Based on a 4-fold or greater reduction of the MIC after the addition of NMP in >50% of the isolates, significant effects of NMP at a concentration of 100 mg/L were seen for levofloxacin, linezolid and ethidium bromide. The ethidium bromide MIC changes after NMP addition correlated with differences in the ethidium bromide intracellular accumulation as measured by fluorometry in whole cell accumulation experiments. The activity of PAbetaN was different from that of NMP, in particular regarding macrolide resistance reversal, suggesting different modes of action of the two putative EPIs. CONCLUSIONS: NMP is moderately active in reversing MDR in clinical isolates of E. coli and can partially restore fluoroquinolone susceptibility through inhibition of efflux pumps.     

Identification of cross-reactive and genuine Parietaria judaica pollen allergens

BACKGROUND: The weed Parietaria judaica is one of the most important pollen allergen sources in the Mediterranean area. OBJECTIVE: We sought to identify P judaica pollen allergen, which might be used to serologically distinguish genuine Parietaria sensitization and cross-reactivity to allergens from other weed species (eg, mugwort and ragweed). METHODS: The allergen profile of P judaica IgE-reactive sera from weed pollen-sensitized allergic individuals from the Mediterranean region (n = 36) with high Parietaria pollen exposure and from weed pollen-allergic patients with little or no Parietaria exposure (Austria, n = 42; Scandinavia, n = 8; United States, n = 19) was established by CAP FEIA measurements and by IgE immunoblot inhibition experiments with recombinant allergens. RESULTS: The majority (83%) of the Mediterranean weed pollen-allergic patients mounted high IgE antibody levels (mean specific IgE, 20.89 kUA/L) against recombinant (r) Par j 2, whereas only 7% of the non-Mediterranean weed-allergic patients showed low IgE reactivity to rPar j 2 (mean specific IgE, 1.03 kUA/L). The cytoskeletal protein profilin and a 2-EF-hand calcium-binding allergen were identified as cross-reactive Parietaria allergens, which were recognized preferentially by Parietaria -positive, non-Mediterranean weed pollen-allergic patients. CONCLUSION: rPar j 2 might be used as a diagnostic marker allergen to identify weed pollen-allergic patients who are genuinely sensitized against Parietaria pollen and thus would be particularly suited for specific immunotherapy with Parietaria pollen extract.     

Roles of the Maltese cross form in the development of parasitemia and protection against Babesia microti infection in mice

Babesia microti, a hemoprotozoan parasite of rodents, is also important as a zoonotic agent of human babesiosis. The Maltese cross form, which consists of four masses in an erythrocyte, is characteristic of the developmental stage of B. microti. Monoclonal antibody (MAb) 2-1E, which specifically recognizes the Maltese cross form of B. microti, has been described previously. In the present study, we examined the roles of the Maltese cross form during the infectious course of B. microti in mice. The number of the Maltese cross form increased in the peripheral blood of infected mice prior to the peak of parasitemia. With confocal laser scanning microscopy, MAb 2-1E was found to be reactive with the ring form, with the parasites undergoing transformation to the Maltese cross form and subsequent division, and also with extracellular merozoites. Furthermore, the Maltese cross form-related antigen (MRA) gene was isolated from a B. microti cDNA library by immunoscreening with MAb 2-1E, and the nucleotide sequence was determined. Genomic analyses indicated that the MRA gene exists as a single-copy gene in B. microti. Immunization of mice with recombinant MRA induced significant protective immunity against B. microti infection. These findings indicate that the Maltese cross form plays important roles in both the development of parasitemia and the protective response against the infection.     

Bridging the gap: how traditional healers interact with their patients. A comparative study in Cameroon

Objective To compare traditional healers (TH) and Cameroonian representatives of Western medicine (Western providers (WP)) in terms of patient characteristics and communication patterns during the consultation in rural Cameroon. Methods A facility‐based comparative study was conducted. Seven TH were compared to eight WP in the same district. Patients (five per provider) provided detailed socio‐demographic data. Recorded consultations were analysed with the Roter Interaction Analysis System (RIAS). Results Patients were similar in socio‐demographic characteristics except for age, where TH patients were on average 9 years younger (P < 0.05). Patients of TH travelled 2.5 times as far to their provider as did patients in the WP group (79 vs. 31 km; P < 0.05) and paid 12 times more for their treatment (123 vs. 10 Euros; P < 0.05). Consultations of TH were shorter (5.6 vs. 10.3 min, P < 0.01), had fewer utterances (100 vs. 166, P < 0.05) and the patient’s share in the communication was smaller (P < 0.01). TH had a higher percentage of lifestyle and psychosocial information at the expense of medical information (P < 0.05) and communicated more emotionally (P < 0.001). They asked more frequently for their patients’ opinion (P < 0.01) and explicitly discussed their patients’ concept of illness (P < 0.001). Patients of TH responded with a higher percentage of active communication (P < 0.05) i.e. question asking (P < 0.01). Conclusions Our data contradict the idea that the lack of money or geographical access to Western health care in rural Africa is the main reason for people to consult traditional healers. Compared to WP, TH interacted very differently with their clients, using a more patient‐centred communication style, to seek common ground with patients. This different type of interaction could be a relevant factor contributing to the popularity of traditional healers in Cameroon.     

Semisynthetic homoharringtonine induces apoptosis via inhibition of protein synthesis and triggers rapid myeloid cell leukemia-1 down-regulation in myeloid leukemia cells

Semisynthetic homoharringtonine (ssHHT) is now being evaluated in phase II clinical trials for the treatment of chronic myelogenous leukemia and acute myelogenous leukemia patients. Here, we examined the mechanism of the apoptosis induced by ssHHT in myeloid leukemia cells. First, we have shown that ssHHT induces apoptosis in HL60 and HL60/MRP cell lines in a time- and dose-dependent manner, and independently of the expression of Bax. The decrease of mitochondrial membrane potential and the release of cytochrome c were observed in the apoptotic cells induced by ssHHT. To unveil the relationship between ssHHT and the mitochondrial disruption, we have shown that ssHHT decreased myeloid cell leukemia-1 (Mcl-1) expression and induced Bcl-2 cleavage in HL60 and HL60/MRP cell lines. The Bcl-2 cleavage could be inhibited by the Z-VAD.fmk caspase inhibitor. However, Mcl-1 turnover was very rapid and occurred before caspase activation. The Mcl-1 turnover was only induced by ssHHT and cycloheximide, but not by daunorubicin and cytosine arabinoside, and could be restored by proteasome inhibitors. Second, we confirmed that ssHHT rapidly induced massive apoptosis in acute myelogenous leukemia patient cells. We have also confirmed the release of cytochrome c and a rapid turnover of Mcl-1 in these patient cells, taking place only in apoptotic cells induced by ssHHT but not in cells undergoing spontaneous apoptosis. Finally, we have shown that ssHHT inhibits protein synthesis in both cell line and patient cells. We suggest that the inhibition of protein synthesis and resulting Mcl-1 turnover play a key role in the apoptosis induced by ssHHT. Our results encourage further clinical trials for the use of ssHHT in acute myelogenous leukemia.     

Structural Reasons for Restricted Backbone Motion in Poly(n‐alkyl methacrylates): Degree of Polymerization,Tacticity and Side‐Chain Length

Summary: The complex dynamics of poly(n‐alkyl methacrylates) is studied by advanced ¹³C NMR spectroscopy as well as mechanical and dielectric relaxation. Extended backbone conformations are identified as the molecular units involved in structural relaxation. From the variation in the degree of polymerization and a comparison with the presence of stereoregular sequences in the sample, the length of the extended units is determined to involve about five, at most ten monomeric units. Syndiotactic and isotactic sequences behave similarly. These findings are indicative of locally structured polymer melts.

     

PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA,a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants

The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.