Potential Role for IL-2 ELISpot in Differentiating Recent and Remote Infection in Tuberculosis Contact Tracing

Interferon (IFN)-γ release assays (IGRA) have improved tuberculosis contact tracing, but discrimination of recent from remote Mycobacterium tuberculosis contacts is not possible by IGRA alone. We present results of a tuberculosis contact investigation with a new early-secretory-antigenic-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 specific interleukin (IL)-2 ELISpot in addition to ESAT-6 and CFP-10 specific IFN-γ ELISpot and tuberculin skin testing (TST). Results of the TST, IFN-γ ELISpot and IL-2 ELISpot were positive in 6/172 (3.4%), 7/167 (4.2%) and 6/196 (3.1%) of contacts, respectively. Close contact (≥100 hours) to the index case increased the risk of positive results in the IFN-γ ELISpot, TST, and IL-2 ELISpot by 40.8, 19.3, and 2.5 times, respectively. Individuals with a positive IFN-γ ELISpot/negative IL-2 ELISpot result had a median (IQR) duration of index case exposure of 568 hours (133_1000) compared to individuals with a positive IFN-γ ELISpot/positive IL-2 ELISpot result (median = 24 hours; 20_130; p-value = 0.047). Combination of a M. tuberculosis specific IFN-γ ELISpot with a M. tuberculosis specific IL-2 ELISpot significantly improved the identification of individuals with the highest risk of recent M. tuberculosis infection and is a promising method that should be explored to target tuberculosis preventive chemotherapy.     

Rheological and structural characterization of the interactions between cyclodextrin compounds and hydrophobically modified alginate

Interactions in semidilute solutions of a hydrophobically modified alginate (HM-alginate) in the presence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) monomer or a beta-cyclodextrin polymer (poly(beta-CD)) have been characterized at different temperatures with the aid of rheology and small-angle neutron scattering (SANS). The viscosity results for the HM-alginate/HP-beta-CD system reveal progressive deactivation of the hydrophobic associations as the concentration of HP-beta-CD increases. For the HM-alginate/poly(beta-CD) system, on the other hand, addition of poly(beta-CD) sets up bridges between adjacent polymer chains and thereby strengthens the associative network. A novel shear-thickening effect is observed at fairly high shear rates for the HM-alginate/poly(beta-CD) system, and this feature is influenced by temperature. Elevated temperature induces higher chain mobility and the formation of weaker network associations. Analyses of the SANS data disclosed that the association strength in HM-alginate/poly(beta-CD) mixtures increases strongly with increasing cosolute concentration, whereas no effect or a moderate weakening of the strength can be traced in HM-alginate/HP-beta-CD solutions upon addition of HP-beta-CD. The value of the correlation length xi is virtually not affected by the addition of cosolute for the HM-alginate/poly(beta-CD) system, whereas the decoupling of hydrophobic moieties of the polymer upon addition of HP-beta-CD gives rise to a smaller value of xi, suggesting that the size of the heterogeneity patches is reduced. The SANS results suggest that compact association structures are formed in the HM-alginate/poly(beta-CD) solutions.     

Head and neck cancer cells are efficiently infected by Ad5/35 hybrid virus

BACKGROUND: Clinical gene therapy trials using standard Ad5-based vectors have thus far demonstrated limited efficacy, most likely due to low expression levels of adenoviral receptors on tumor cells. We sought to analyze adenoviral receptor expression levels on primary head and neck squamous cell carcinoma (HNSCC) cells and to determine whether adenoviral retargeting to the CD46 receptor via the Ad5/35 system would increase therapeutic potential for HNSCC. METHODS: We used flow cytometric analyses to determine adenoviral receptor expression levels on nine primary HNSCC cells collected from cancer patients. Adenoviruses Ad5.LacZ and Ad5/35.LacZ were used to analyze the differences in viral transduction both in vitro and in a HNSCC tumor mouse model. RESULTS: Flow cytometric analyses demonstrated uniformly high CD46 expression in all cells studied (85-99%). In contrast, coxsackievirus and adenovirus receptor (CAR) expression was substantially lower and highly variable (1.6-62%). Alpha(v) integrin expression was between 39-98%. In situ stainings for beta-galactosidase gene expression demonstrated that Ad5/35.LacZ was clearly more effective than Ad5.LacZ in transducing primary HNSCC cells. Quantification of beta-galactosidase expression revealed up to 65 times higher transgene expression from Ad5/35.LacZ than Ad5.LacZ. In vivo, beta-galactosidase expression was detected in a substantial area after a single intratumoral injection of Ad5/35.LacZ, whereas injection with Ad5.LacZ resulted in gene expression only in a few cells. CONCLUSIONS: Our results demonstrate that the low and variable CAR expression levels limit the therapeutic efficacy of Ad5-based strategies for HNSCC. In contrast, the effective in vivo transduction capacity of Ad5/35 warrants further development of this vector for the treatment of head and neck cancer.     

Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: review and new insights from an "inverse" deuterium isotope effect

We review our work on electron transfer and proton dynamics during photoactivation in DNA photolyase from E. coli and discuss a recent theoretical study on this issue. In addition, we present unpublished data on the charge recombination between the fully reduced FADH(-) and the neutral (deprotonated) radical of the solvent exposed tryptophan W306. We found a pronounced acceleration with decreasing pH and an inverse deuterium isotope effect (k(H)/k(D)=0.35 at pL 6.5) and interpret it in a model of a fast protonation equilibrium for the W306 radical. Due to this fast equilibrium, two parallel recombination channels contribute differently at different pH values: one where reprotonation of the W306 radical is followed by electron transfer from FADH(-) (electron transfer time constant tau(et) in the order of 10-50 micros), and one where electron transfer from FADH(-) (tau(et)=25 ms) is followed by reprotonation of the W306 anion.     

The structure of tri-proline in water probed by polarized Raman, Fourier transform infrared, vibrational circular dichroism, and electric ultraviolet circular dichroism spectroscopy

Tripeptidesserve as model systems for understanding the so-called random-coil state of peptides and proteins. While it is well known that polyproline or proline-rich polypeptides adopt the very regular polyproline-II (PPII) or left-handed 3(1)-helix conformation, it was thus far not clear whether this is also the predominant structure adopted by proline-containing tripeptides. To clarify this issue, we have investigated the amide I' band profile in the ir, isotropic, and anisotropic Raman, and vibrational circular dichroism (VCD) spectrum of cationic and zwitterionic tri-proline in D(2)O. The data were analyzed by modifying a recently developed algorithm, which allows one to obtain the central dihedral angles of tripeptides from the amide I' band intensities (R. Schweitzer-Stenner, Biophysical Journal, 2002, Vol. 83, pp. 523-532). Our analysis revealed that the peptide adopts a nearly canonical PPII structure in water with psi and phi values in the range of 175 degrees -165 degrees and -70 degrees -(-80 degrees ), respectively. This is fully confirmed by the respective electronic ultraviolet-CD spectra. Our result indicates that the strong PPII propensity of trans proline results from local interactions between the pyrrolidine ring and the backbone and is not due to any long-range interactions.     

Evaluation of a new fiber-optic pressure recording system for cardiovascular measurements in mice

We have tested a new fiber-optic pressure recording system, Samba, with a thin fiber [outer diameter (OD) = 0.25 mm] and a pressure sensor (length and OD = 0.42 mm) attached to the end. The accuracy of the system tested in vitro was good, with a coefficient of variation of 2.54% at 100 mmHg. The drift was <0.45 mmHg/h, and the temperature sensitivity was approximately 0.07 mmHg/1 degrees C between 22 and 37 degrees C. The frequency response characteristics were similar to a 1.4-Fr Millar catheter (0-200 Hz). Introduction of the Samba sensor from the right carotid artery into the left ventricle in six mice caused no drop in mean aortic pressure, whereas introduction of a 1.4-Fr Millar catheter (OD = 0.47 mm; n = 6) caused a pressure drop from 91.6 +/- 9.2 to 65.1 +/- 6.2 mmHg; P < 0.05. Thus the Samba sensor system may represent a new alternative to assess hemodynamic variables in the murine cardiovascular system.     

Relationship of the Xeroderma Pigmentosum Group E DNA Repair Defect to the Chromatin and DNA Binding Proteins UV-DDB and Replication Protein A

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB⁻ XP-E cell extracts, but microinjection of the protein into DDB⁻ XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.The heritable human disorder xeroderma pigmentosum (XP) is chiefly characterized by an increased incidence of benign and malignant skin lesions after exposure to sunlight. Affected individuals fall into one of eight different genetic complementation groups. Cells from the seven complementation groups A through G have reduced nucleotide excision repair (NER) of damaged DNA, while cells from the variant, or V, group are defective in a less-defined process of cellular recovery after DNA damage (11). Genes and proteins representing XP groups A (XP-A) B, C, D, F, and G have all been isolated and found to represent some of the subunits of the core NER recognition and incision machinery. XP-E is the mildest form of the disorder, and cells of this group generally have 40 to 60% of the normal repair level, as shown by autoradiographic measurement of unscheduled DNA synthesis (UDS) after UV irradiation. Cell fusion studies have assigned at least 16 individuals to this form of the disorder (6, 19, 23, 40).There are several indications that a DNA damage binding protein denoted UV-DDB (or DDB) is involved in the primary XP-E defect. The protein has been detected in extracts of vertebrate cells as an activity that preferentially binds damaged oligonucleotides in electrophoretic mobility shift or filter binding assays. The protein has a particular affinity for (6-4) photoproducts in UV-irradiated DNA (10, 15, 16, 34, 41, 43), but UV-DDB also binds to DNA damaged by other agents, including cisplatin and nitrogen mustard (32). The activity has been purified as a single 127-kDa protein (2) and as a complex with two subunits of 127 and 48 kDa (21). Damage-binding activity is missing from some cells in the XP-E group, designated DDB⁻, but is present in other XP-E cell lines, designated DDB⁺ (3, 15, 19, 23). The genes encoding the p127 protein (7, 17, 39) and the p48 protein (7) have been isolated, but DNA sequence features have not yet yielded firm clues about their functions. Microinjection of purified UV-DDB into XP-E cells lacking UV-DDB activity substantially corrects the NER defect, as measured by UDS after UV irradiation, but UV-DDB⁺ cells are not corrected (22). Sequence alterations in the gene for p48 have been reported for several XP-E cell lines (29), and it is possible that these are causative mutations for XP-E.There are also suggestions that the single-stranded DNA binding activity of replication protein A (RPA) is involved in the XP-E defect. RPA is a heterotrimer of three subunits with sizes of 70, 34, and 14 kDa that plays key roles in DNA replication, recombination, and DNA repair (44). It is one of the core components of the eukaryotic nucleotide excision-incision system (1, 12, 28). With regard to XP, it was recently reported that XP-E cell extracts are severely defective in NER in vitro and that RPA can specifically correct the repair defect of these extracts, but not those of extracts of other complementation groups (20). Moreover, it has been found that RPA copurifies to some extent with UV-DDB protein and that the two proteins interact, showing a tighter association with chromatin after UV irradiation of cells (31).The availability of lymphoblastoid cell lines derived from three newly identified XP-E individuals has given us the opportunity to further investigate the possible relationships of UV-DDB and RPA to the molecular defect in XP-E and the influence of these proteins on NER.     

Snail-dependent and -independent epithelial-mesenchymal transition in oral squamous carcinoma cells.

Disappearance of E-cadherin is a milestone for epithelial-mesenchymal transition (EMT), found both in carcinomas and in some fibrotic diseases. We have studied the mechanisms of EMT in oral squamous cell carcinoma (SCC) cells isolated from primary tumor (43A) and its recurrent tumor (43B). Whereas the cells from primary carcinoma displayed a typical phenotype of squamous epithelial cells including E-cadherin and laminin-332 (laminin-5), cells from recurrent tumor expressed characteristics of dedifferentiated, EMT-experienced tumors. 43B cells expressed E-cadherin repressors ZEB-1/deltaEF1 and especially ZEB-2/SIP1, which therefore appear as candidates for endogenous EMT in these cells. Differences between endogenous and exogenous EMT were assessed by transfecting 43A cells with SNAIL cDNA. SNAIL-transfected cells showed complete EMT phenotype with fibroblastoid appearance, vimentin filaments, E-cadherin/N-cadherin switch, lack of hemidesmosomes and, as a new feature of EMT, lack of laminin-332 synthesis. Upregulation of ZEB-1 and ZEB-2 was evident in these cells, suggesting that SNAIL can regulate these E-cadherin repressors. New monoclonal antibodies against SNAIL showed nuclear immunoreactivity not only in the SNAIL-transfected cells but also in carcinoma cells lacking production of Lm-332 and showing signs of EMT. These results suggest that changes in the epithelial cell differentiation program and EMT in SCC cells can result from the interplay among several E-cadherin repressors; however, SNAIL alone is able to accomplish a complete EMT.     

Tetranectin Is a Novel Marker for Myogenesis during Embryonic Development, Muscle Regeneration, and Muscle Cell Differentiationin Vitro

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiationin vitro.We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophicmdxmice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiationin vitro.Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.     

Leptomeric fibrils and T-tubule desmosomes in the Z-band region of the mouse heart papillary muscle

Summary The papillary muscle of the heart of adult white mice is investigated. Intrafibrillarly located leptomeric fibrils, frequently encountered in the Z-band region of the myofibrils. The leptomeric fibrils are always running in a transverse direction and often in close proximity to the transverse tubules (which are also located at this level). There seems to be a close connection between the dense striae of the leptofibrils and the Z-bands of ordinary myofibrils. The leptomeric fibrils are spindle-shaped and have a length varying between 0.6 and 1.2 m. The banding periodicity of the fibrils is approximately 0.16 m.Occasionally desmosomes are observed in the T-tubule system.