Estimating diagnostic accuracy of tests for latent tuberculosis infection without a gold standard among healthcare workers.

The evaluation of diagnostic accuracy of new in vitro diagnostic assays for tuberculosis infection has been hampered by the lack of a standard reference test. The aim of this study was to compare sensitivity and specificity of interferon gamma assays for latent tuberculosis infection by assessing the association of test results with tuberculosis occupational exposure and by using latent class analysis. We analysed data from 115 healthcare workers on whom tuberculin skin test (TST) and the following in vitro tests were performed: in-house ELISPOT for RD1 proteins, T.SPOT-TB and Quantiferon-TB Gold. Results of all tests were associated with increased occupational risk of exposure to Mycobacterium tuberculosis, but only TST was associated with Bacillus Calmette-Guerin (BCG) vaccination. Sensitivity/specificity (95% confidence intervals) estimated by a latent class model were: 99.9%/64.2% (53.0-74.1) for TST, 95.3% (61.8-99.6)/87.5% (78.0-93.2) for in-house ELISPOT, 96.7% (69.3-99.7)/85.6%(75.3-92.0) for T.SPOT-TB, and 76.3% (55.9-89.1)/93.6% (85.4-97.3) for Quantiferon. The estimated specificity of in vitro assays was higher than that of TST also among individuals who were not BCG-vaccinated. In conclusion, when used in healthcare workers, in vitro assays may provide a significant increase of specificity for tuberculosis infection compared to TST, even among non vaccinated individuals, at the cost of some sensitivity.

EBOLA Ag K-SeT rapid test: field evaluation in Sierra Leone.

OBJECTIVES: Efficient interruption of Ebola virus disease (EVD) transmission chains critically depends on reliable and fast laboratory diagnosis. We evaluated the performance of the EBOLA Virus Antigen Detection K-SeT (EBOLA Ag K-SeT), a new rapid diagnostic antigen test in field settings. METHODS: The study was conducted in a field laboratory located in Freetown (Sierra Leone) by the Italian National Institute for Infectious Diseases 'L. Spallanzani' and the EMERGENCY Onlus NGO. The EBOLA Ag K-SeT was tested on 210 residual plasma samples (EVD prevalence 50%) from patients hospitalized at the EMERGENCY Ebola treatment center in Goderich (Freetown), comparing the results with quantitative real-time PCR. RESULTS: Overall, the sensitivity of EBOLA Ag K-SeT was 88.6% (95% confidence interval (CI), 82.5-94.7), and the corresponding specificity was 98.1% (95% CI, 95.5-100.7). The positive and negative predictive values were 97.9% (95% CI, 95.0-100.8) and 89.6% (95% CI, 84-95.2), respectively. The sensitivity strongly increased up to 98.7% (95% CI, 96.1-101.2) for those samples with high virus load (≥6.2 log RNA copies/mL). CONCLUSIONS: Our results suggest that EBOLA Ag K-SeT could represent a new effective diagnostic tool for EVD, meeting a need for resource-poor settings and rapid diagnosis for individuals with suspected EVD.

Autologous hematopoietic stem cell transplantation has better outcomes than conventional therapies in patients with rapidly progressive systemic sclerosis.

We retrospectively evaluated the efficacy of autologous hematopoietic stem cell transplantation (AHSCT) in 18 patients with rapidly progressive diffuse cutaneous systemic sclerosis (rp-dcSSc), and compared their disease outcomes with those of 36 demographically- and clinically-matched patients treated with conventional therapies. Cutaneous involvement, by performing modified Rodnan skin score (mRss), lung diffusion capacity, by measuring diffusing capacity of lung for carbon monoxide (DLCO), and disease activity, by applying the European Scleroderma Study Group (ESSG) scoring system, were the outcome variables measured at the baseline time and then every 12 months for the following 60 months in both the AHSCT-treated patients and the control group. In the AHSCT group, treatment-related mortality was 5.6%. In this group, both mRss and ESSG scores showed a significant reduction 1 year after AHSCT (P<0.002); and these results were maintained until the end of follow-up. Conversely, DLCO values remained stable during the whole period of follow-up. Survival rate of AHSCT group was much higher than that observed in the whole control group (P=0.0005). The probability that the ESSG score and mRss would remain at a high level, and DLCO could decrease, was significantly higher in the control group as a whole and in the subgroup of control patients treated with cyclophosphamide than in the AHSCT group. This study confirms that the AHSCT is effective in prolonging survival, as well as in inducing a rapid reduction of skin involvement and disease activity, and preserving lung function in patients with rp-dcSSc.

Accuracy of an immune diagnostic assay based on RD1 selected epitopes for active tuberculosis in a clinical setting: a pilot study.

A previous case-control study reported that an in-vitro interferon (IFN)-gamma response to early secreted antigenic target (ESAT)-6 selected peptides was associated with active tuberculosis (A-TB). The objective of the present pilot study was to evaluate the diagnostic accuracy of this assay for TB disease in a clinical setting. An IFN-gamma ELISPOT assay was performed on samples from patients with suspected A-TB using two peptides selected from ESAT-6 protein and three peptides selected from culture filtrate 10 (CFP-10) proteins. The results were compared with those obtained by two commercially available assays approved for diagnosis of TB infection (T SPOT-TB and QuantiFERON-TB Gold) which use ESAT-6/CFP-10 (RD1) overlapping peptides. Sensitivity to the RD1 selected peptides was 70% (positive for 16 of 23 patients with microbiologically diagnosed A-TB) and specificity was 91% (positive for three of 32 controls). In contrast, the sensitivity and specificity were 91% and 59%, respectively, for T SPOT-TB, and were 83% and 59%, respectively, for QuantiFERON-TB Gold. The RD1 selected peptides assay had the highest diagnostic odds ratio for A-TB. Thus, the results suggest that an assay based on RD1 selected peptides has a higher diagnostic accuracy for A-TB in a clinical setting compared with commercially available assays based on RD1 overlapping peptides.

In vitro activity of human chorionic gonadotropin (hCG)--doxorubicin conjugates against ovarian cancer cells.

BACKGROUND: Despite radical surgery and the introduction of novel chemotherapeutic agents, the prognosis of ovarian cancer remains poor. Since in the past the potential role of gonadotropins in the induction and progression of ovarian cancer has been discussed, we conjugated doxorubicin to human chorionic gonadotropin (hCG) in order to specifically target ovarian cancer cells. MATERIALS AND METHODS: Doxorubicin was conjugated to hCG via glutaraldehyde. 48 hours post seeding, NIH:OVCAR 3 cells were treated with various concentrations of hCG-conjugated and non-conjugated doxorubicin for 2 hours. Cells were cultured for a total of 168 hours. Cell growth was monitored by a crystal violet assay. RESULTS: Conjugating doxorubicin to hCG resulted in an average specific uptake of 22 mol doxorubicin per mol protein (range 3.0-43.3 mol). Incubating NIH:OVCAR 3 cells for 2 hours with the conjugate led to a more than 8 fold increase of the IC50 values compared to non-conjugated doxorubicin (0.55 microM versus 4.43 microM). The antiproliferative activity of both conjugated and free doxorubicin was detectable up to 168 hours post treatment. CONCLUSIONS: The present experiments clearly demonstrate a more than 8-fold increase in cytotoxicity of the conjugates compared to free doxorubicin. It was also shown that this effect was not restricted to an acute toxic event but that it resulted in a prolonged antiproliferative activity.

Different cytokine production and effector/memory dynamics of alpha beta+ or gamma delta+ T-cell subsets in the peripheral blood of patients with active pulmonary tuberculosis.

Immunity to M.tuberculosis (MTB) infection consists of interactions between various T-cell subsets that control the infection and prevent further reactivation. We analysed the effector/memory T-cell dynamics and cytokines production in the peripheral blood of patients with pulmonary tuberculosis (TB). We observed that the frequency of CD4+ T-cell effectors was significantly increased during active TB, confirming a major role of this T-cell subset in TB immunity. Pre-terminally differentiated CD8+ T-lymphocytes were increased in the peripheral blood as well. In contrast, we observed a reduced number of effector mycobacteria-reactive gammadelta+ T-lymphocytes with a specific defects in reacting to mycobacterial nonpeptidic antigens, suggesting that this innate response is rapidly lost during TB infection. Nevertheless, the frequency of gammadelta+ T-cells effectors in TB patients was higher than the alphabeta+ T-cell response to peptide from MTB-ESAT-6 protein and quantitatively similar to PPD reactivity. Thus, alphabeta+ and gammadelta+ T-cell differentiation and function are differently triggered by active TB infection.

Bendamustine in combination with ofatumumab in relapsed or refractory chronic lymphocytic leukemia: a GIMEMA Multicenter Phase II Trial.

We conducted a phase II, noncomparative, open-label, multicenter GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) study (CLL0809) to assess the efficacy and safety of bendamustine in combination with ofatumumab (BendOfa) in relapsed/refractory chronic lymphocytic leukemia (CLL). Forty-seven patients from 14 centers were evaluated. Therapy consisted of bendamustine (70 mg/m(2)) for 2 consecutive days every 28 days, and ofatumumab 300 mg on day 1 and 1000 mg on day 8 during the first cycle, and 1000 mg on day 1 subsequently. Treatment was administered up to six cycles. The overall response rate (ORR), as per intention-to-treat analysis, was 72.3% (95% confidence of interval (CI), 57-84%), with 17% complete responses. After a median follow-up of 24.2 months, the overall survival was 83.6% (95% CI, 73.0-95.7%) and the progression-free survival (PFS) was 49.6% (95% CI, 35.9-68.6%). The median PFS was 23.6 months. Univariate and multivariate analyses were used to identify clinical and biological characteristics associated with ORR and PFS. Myelosuppression was the most common toxicity; grade ≥3 neutropenia was observed in 61.7% of patients; however, grade ≥3 infections occurred in 6% of patients. BendOfa is feasible and effective in relapsed/refractory CLL patients, including patients with high-risk clinical and biological features.

Response to region of difference 1 (RD1) epitopes in human immunodeficiency virus (HIV)-infected individuals enrolled with suspected active tuberculosis: a pilot study.

Tuberculosis is the most frequent co-infection in human immunodeficiency virus (HIV)-infected individuals, and which still presents diagnostic difficulties. Recently we set up an assay based on interferon (IFN)-gamma response to region of difference 1 (RD1) peptides selected by computational analysis which is associated with active Mycobacterium tuberculosis replication. The objective of this study was to investigate the response to RD1 selected peptides in HIV-1-infected individuals in a clinical setting. The mechanisms of this immune response and comparison with other immune assays were also investigated. A total of 111 HIV-infected individuals with symptoms and signs consistent with active tuberculosis were enrolled prospectively. Interferon (IFN)-gamma responses to RD1 selected peptides and recall antigens were evaluated by enzyme-linked immunospot assay. Results were correlated with CD4(+) T cell counts, individuals' characteristics, tuberculin skin test, QuantiFERON-TB Gold and T-SPOT.TB. Results from 21 (19%) individuals were indeterminate due to in vitro cell anergy. Among 'non-anergic' individuals, sensitivity for active tuberculosis of the assay based on RD1 selected peptides was 67% (24 of 36), specificity was 94% (three of 54). The assay also resulted positive in cases of extra-pulmonary and smear-negative pulmonary active tuberculosis. The response was mediated by CD4(+) effector/memory T cells and correlated with CD4(+) T cell counts, but not with plasma HIV-RNA load. Moreover, the RD1 selected peptides assay had the highest diagnostic odds ratio for active tuberculosis compared to tuberculin skin test (TST), QuantiFERON-TB Gold and T-SPOT.TB. RD1 selected peptides assay is associated with M. tuberculosis replication in HIV-infected individuals, although T cell anergy remains an important obstacle to be overcome before the test can be proposed as a diagnostic tool.

Retinoic acid and thyroid hormone may function through similar and competitive pathways in regenerating axolotls.

The objective of this study was to determine whether thyroid hormone (TH) would interfere with retinoic acid (RA), which proximalizes axolotl larvae regenerate limb pattern. RA and TH are ligands for members of the steroid hormone thyroid hormone nuclear binding protein superfamily which form functional homodimers, but may also form stable heterodimers with the RXR protein and may recognize identical DNA sequences. TH alone does not affect limb pattern but induces metamorphosis in regenerating animals. Coinjected animals do not metamorphose, and when compared to RA controls regenerate more proximal and in some cases anteroposterior (AP) and dorsoventral (DV) duplicate limb structures. In addition, the tissues that are normally lost or changed during metamorphosis appear to be sensitized resulting in the formation of (1) new dorsal gill lamellae accompanied by bifurcation and broadening of the original gill lamellae, (2) partial resorption of the tail fin, and (3) changes in eye position and snout morphology. Bifurcation of gill lamellae tips, but not the formation of supernumerary gills, is also observed in animals treated with RA alone. These results indicate that the molecular mechanism of RA and TH function through similar and perhaps competitive pathways.

Structural organization and stability of a thermoresistant domain generated by in vivo hydrolysis of the alpha-crystallin B chain from calf lens.

A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro cross-linking of calf lens alpha-crystallin by malondialdehyde.

The effect of malondialdehyde on structural features of bovine alpha-crystallin has been investigated by absorption and fluorescence spectroscopy as well as by far-UV circular dichroism. Experimental evidence suggests the occurrence of intermolecular cross-linking induced by malondialdehyde. This cross-linking does not seem to affect the tryptophan environment, as suggested by intrinsic protein fluorescence. On the contrary, the time dependence of far-UV dichroic activity indicates that the cross-linking is accompanied by a secondary structure change. The formation of high molecular mass aggregates, evidence by electrophoresis in denaturing conditions, leads to irreversible alpha-crystallin aggregation due to extensive intermolecular cross-linking. Since malondialdehyde is produced in vivo as a breakdown product of lipid peroxidation the possible involvement of this molecule in the pathological mechanism of cataract formation has been briefly discussed.

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins.

Primary structure analysis of the four river buffalo alpha-globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two alpha-globin chains, Ialpha1 and IIalpha3, which differ at positions 129 and 131: Ialpha1 has 64 Ala, 129 Phe, 131 Asn; IIalpha3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two alpha-globin chains, Ialpha2 and IIalpha4, which differ at positions 10 and 11: Ialpha2 has 10 I1e, 11 Gln, 64 Asn; IIalpha4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic alpha-globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo alpha-globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.