Construction, training and clinical validation of an interpretation system for genotypic HIV-1 drug resistance based on fuzzy rules revised by virological outcomes

OBJECTIVES: To evaluate whether fuzzy operators can be usefully applied to the interpretation of genotypic HIV-1 drug resistance by experts, and to improve the prediction of salvage therapy outcome by adapting interpretation rules of genotypic resistance on the basis of their association with virological response data. METHODS: We used a clinical dataset of 231 patients failing highly active antiretroviral therapy (HAART) and starting salvage therapy with baseline resistance genotyping and virological outcomes after 3 and 6 months. A set of rules predicting genotypic resistance was initially derived from an expert (ADL). Rules were implemented using a fuzzy logic approach and the virological outcomes dataset used for the training phase. The resulting algorithm was validated using a separate set of 184 selected patients by correlating the resulting predicted activity with observed virological response at 3 months. For comparison, the expert systems from the drug resistance group of the Agence Nationale de Recherches sur le SIDA (ANRS-AC11) and the algorithm from the Stanford's HIV drug resistance database (Stanford HIVdb) were evaluated on the same set. RESULTS: The starting algorithm had a correlation with virological outcomes of R2=0.06 (P=0.0001). After the training phase the correlation with virological outcomes increased to R2=0.19 (P<0.000001). In the validation set of patients, the activity of the salvage regimen predicted by the fuzzy algorithm was the only variable independently predictive of the 3-month viral load change even after adjusting by the activity predicted by the two expert systems and baseline viral load (for each 10% salvage regimen's activity increase, mean HIV RNA change from baseline: -0.27 log10 copies/ml; 95% CI -0.39, -0.15). CONCLUSION: Using fuzzy operators in a virological outcomes training database to implement a rules-based algorithm for genotypic resistance interpretation, significant improvements of outcomes prediction were obtained. The resulting algorithm showed an independent predictive capability of virological outcomes over that of two rules-based interpretation algorithms made by experts. Although the system was trained and validated on a limited number of cases, the approach deserves further evaluation.     

The length of treatment of aggressive non-Hodgkin's lymphomas established according to the international prognostic index score: long-term results of the GISL LA03 study

OBJECTIVES: To compare two different schedules of two different anthracycline-containing regimens, where length of treatment is modulated according to the international prognostic index (IPI) in patients with aggressive non-Hodgkin's Lymphoma (NHL). METHODS: In 1993 the Gruppo Italiano per lo Studio dei Linfomi (GISL) started a randomized 2 x 2 factorial phase III clinical trial for patients with newly diagnosed aggressive NHL comparing ProME(Epidoxorubicin)CE-CytaBOM (PE-C) to ProMI(Idarubicin)CE-CytaBOM (PI-C) and a fixed to a flexible treatment schedule where anthracycline dose was to be modulated according to observed hematological toxicity. Patients with low or low-intermediate IPI (IPI 0-2) and those with intermediate-high or high IPI (IPI 3-5) should receive six or eight courses, respectively. Involved-field radiotherapy was allowed for patients with initial bulky disease or with residual masses. RESULTS: Three hundred and fifty-six patients were registered into the study and randomized. Patients were well balanced among the four study arms in terms of clinical characteristics and prognostic factors. Three hundred and forty-five patients were available for evaluation of study endpoints. At the end of induction therapy complete remission rate was 61%, 5-year failure-free survival (FFS) rate was 40% and 5-year overall survival (OS) rate was 59%; no differences were observed according to treatment arms. Patients in the flexible arm received higher dose intensity of anthracycline (P < 0.001) with no apparent increase in toxicity. However, the flexible schedule was not superior to the fixed one. Patients with IPI 3-5 showed lower response rates (45% vs. 67%: P < 0.0001) and lower 5-year FFS (29% vs. 45%: P < 0.0001) compared to those with IPI 0-2. CONCLUSIONS: six courses of fixed or flexible PE-C or PI-C can determine a promising success rate in patients with advanced aggressive NHL with IPI 0-2, whereas the same regimens are less effective in patients with IPI 3-5, even if two additional courses are delivered. For the latter group of patients innovative approaches are warranted.     

Molecular events underlying interleukin‐6 independence in a subclone of the CMA‐03 multiple myeloma cell line

We explored the molecular mechanisms involved in the establishement of CMA‐03/06, an IL‐6‐independent variant of the multiple myeloma cell line CMA‐03 previously generated in our Institution. CMA‐03/06 cells grow in the absence of IL‐6 with a doubling time comparable with that of CMA‐03 cells; neither the addition of IL6 (IL‐6) to the culture medium nor co‐culture with multipotent mesenchymal stromal cells increases the proliferation rate, although they maintain the responsiveness to IL‐6 stimulation as demonstrated by STAT1, STAT3, and STAT5 induction. IL‐6 independence of CMA‐03/06 cells is not apparently due to the development of an autocrine IL‐6 loop, nor to the observed moderate constitutive activation of STAT5 and STAT3, since STAT3 silencing does not affect cell viability or proliferation. When compared to the parental cell line, CMA‐03/06 cells showed an activated pattern of the NF‐κB pathway. This finding is supported by gene expression profiling (GEP) analysis identifying an appreciable fraction of modulated genes (28/308) in the CMA‐03/06 subclone reported to be involved in this pathway. Furthermore, although more resistant to apoptotic stimuli compared to the parental cell line, CMA‐03/06 cells display a higher sensibility to NF‐κB inhibition induced by bortezomib. Finally, GEP analysis suggests an involvement of a number of cytokines, which might contribute to IL‐6 independence of CMA‐03/06 by stimulating growth and antiapoptotic processes. In conclusion, the parental cell‐line CMA‐03 and its variant CMA‐03/06 represent a suitable model to further investigate molecular mechanisms involved in the IL‐6‐independent growth of myeloma cells. © 2013 Wiley Periodicals, Inc.     

Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric- directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non- homologous interchromosomal exchange.      

The influence of alumina and ultra-high molecular weight polyethylene particles on osteoblast-osteoclast cooperation

Particle-induced macrophage activation, mainly by UHMWPE wear, has been recognized as the biological mechanism leading to periprosthetic bone resorption, which is responsible for the loosening of the total hip replacements (THR). Ceramic-on-ceramic implants have been advocated as a means of reducing wear products. Many studies investigated the effect of alumina (Al(2)O(3)) particles on monocytes/macrophages, but only limited information are available on their participation to bone turnover. An in vitro model was performed to investigate how Al(2)O(3) and UHMWPE particles may influence the osteoblast-osteoclast interaction: human osteoblasts (HOB) were obtained from trabecular bone, while osteoclasts were derived from peripheral blood mononuclear cells (PBMC) of healthy donors. The amount of IL6, TNF alpha, GM-CSF, and other factors acting on the bone turnover, i.e. the 'receptor activator of NF kappa B' ligand (RANKL) and osteoprotegerin (OPG), was detected in culture medium of particle-challenged HOB (HOB-CM). The Al(2)O(3) and UHMWPE particles did not affect either cell viability or TNF and GM-CSF release, while the increase in IL6 release seemed to be dependent on the particle concentration. UHMWPE increased the release of RANKL from HOB, while OPG and OPG-to-RANKL ratio were significantly inhibited. The ability of HOB-CM to promote osteoclastogenesis was tested via osteoblast/monocyte cooperation: after seven days of culture UHMWPE HOB-CM induced a large amount of multinucleated TRAP-positive giant cells, as well as significantly reduced the amount of IL6, GM-CSF and RANKL in the supernatant. With regard to the inductive effect on the osteoclastogenesis, our results show that the Al(2)O(3) wear debris are less active.     

A one-year follow-up on the effects of raloxifene on thyroid function in postmenopausal women

OBJECTIVE: Estrogens increase serum thyroxine-binding globulin (TBG) and total thyroxine (TT4) concentrations. Serum free thyroxine (FT4) concentrations, however, remain normal. Raloxifene (RAL) is a selective estrogen receptor modulator used to treat postmenopausal osteoporosis. Data on the long-term effects of RAL on thyroid physiology are scanty. We evaluated the effects of RAL administration for 1 year on thyroid function in osteopenic, postmenopausal women. DESIGN: Fifty osteopenic, postmenopausal women were randomly assigned to receive either RAL (60 mg/day, n = 25) or placebo (PL, n = 25) for 1 year, in a double-blind study. Measurements of serum TBG, TT4, FT4, thyroid-stimulating hormone (TSH), thyroid hormone-binding ratio (THBR), FT4 index (FT4-I) and TT4/TBG ratio were carried out at baseline and after 4 and 12 months of therapy. RESULTS: Baseline values were similar in both treatment groups. Serum TBG concentrations were increased during RAL treatment from baseline values of 29.60 +/- 0.9 microg/mL to 31.45 +/- 1.33 and 32.34 +/- 1.37 microg/mL at 4 months and 1 year, respectively (P < 0.05, baseline v 1-year values) but were unchanged during PL treatment. A small, insignificant increase in TT4 and TSH concentrations occurred in the RAL group and no changes in the PL group. All other values were unchanged during either treatment. CONCLUSIONS: These results demonstrate that RAL significantly increased serum TBG levels, but the changes were small and not accompanied by changes in FT4-I, FT4, or TSH concentrations, suggesting that long-term RAL treatment is unlikely to clinically affect the thyroid status in euthyroid, postmenopausal women.     

A prospective, randomised, controlled trial using a Mg-hydroxyapatite - demineralized bone matrix nanocomposite in tibial osteotomy

We in vivo investigated the bone healing ability of a nanocomposite (DBSint®), constituted by biomimetic nano-structured Mg-hydroxyapatite (SINTlife®) and human demineralized bone matrix. Thirty-one subjects undergoing high tibial osteotomy for genu varus were randomly assigned to three groups: during surgery, DBSint® was inserted into nine patients, SINTlife® in thirteen patients and lyophilised bone chips, that is the routine surgery, in nine subjects. As outcome measures, clinical, radiographic and histomorphometry scores were calculated. The osseointegration was evaluated by imaging six weeks, three, six and twelve months after surgery. At six-week follow-up, DBSint® showed a significantly higher osseointegration rate in comparison with lyophilised bone chips (p = 0.008). At the same follow-up, CT-guided bone biopsies were obtained and analysed by histomorphometry: a good osteogenetic potential was demonstrated with DBSint®, as well as with SINTlife® and controls. Unresorbed material was evident with DBSint® and SINTlife®, with a significantly higher percentage in SINTlife® group. At 1-year follow-up, DBSint® was demonstrated as effective and safe as SINTlife® and lyophilized bone chips. More significant results could be obtained by continuing the clinical trial, by increasing the patient number and the study power. Eventually, the role of non-resorbed graft remnants is still unclear and requires further investigation.     

Normal human IgG prevents endothelial cell activation induced by TNFalpha and oxidized low-density lipoprotein atherogenic stimuli

Normal human immunoglobulin G (IgG) has anti-inflammatory and immuno-regulatory properties, which are exploited in the therapy of selected diseases. A putative mechanisms of action is the direct regulation of endothelial cell function by natural antiendothelial cell antibodies. Endothelium activation is a critical event in atherosclerosis. We have verified the ability of normal human IgG to modulate endothelial responses to the atherogenic stimuli tumour necrosis factor-alpha (TNFalpha) and oxidized low-density lipoproteins (oxLDL) in vitro. Confocal microscopy was used to visualize vascular cell adhesion molecule-1 (CD106) expression on endothelial cells, cytoplasmic free calcium ([Ca++]i) modifications and fluorescein-coupled oxLDL internalization. Cytokine secretion was measured by ELISA on cell supernatants. IgG prevented TNFalpha induced CD106 membrane expression and an increase in [Ca++]i, and inhibited the secretion of interleukin-6 (IL-6) and macrophage-colony-stimulating factor (M-CSF). IgG also inhibited CD106 expression induced by oxLDL and one pathway of their internalization, but were ineffective on oxLDL induced [Ca++]i rise and apoptosis. F(ab)'2 fragments from IgG, but not monoclonal IgG, reproduce IgG effects. These findings point to a regulatory role for specific antibodies included in circulating normal IgG towards proinflammatory responses of endothelial cells in atherogenesis and suggest possible development of new therapeutic strategies.     

A 12-Mb Complete Coverage BAC Contig Map in Human Chromosome 16p13.1–p11.2

We have constructed a complete coverage BAC contig map that spans a 12-Mb genomic segment in the human chromosome 16p13.1-p11.2 region. The map consists of 68 previously mapped STSs and 289 BAC clones, 51 of which-corresponding to a total of 7.721 Mb of genomic DNA-have been sequenced, and provides a high resolution physical map of the region. Contigs were initially built based mainly on the analysis of STS contents and restriction fingerprint patterns of the clones. To close the gaps, probes derived from BAC clone ends were used to screen deeper BAC libraries. Clone end sequence data obtained from chromosome 16-specific BACs, as well as from public databases, were used for the identification of BACs that overlap with fully sequenced BACs by means of sequence match. This approach allowed precise alignment of clone overlaps in addition to restriction fingerprint comparison. A freehand contig drawing software tool was developed and used to manage the map data graphically and generate a real scale physical map. The map we present here is approximately 3.5 x deep and provides a minimal tiling path that covers the region in an array of contigous, overlapping BACs.     

Effects of neridronic acid on osteoclasts derived by physiological dual-cell cultures

Increased osteoclastic activity is observed in many osteopathic disorders - including postmenopausal osteoporosis, Paget's disease, primary bone tumours, lytic bone metastases, multiple myeloma and rheumatoid arthritis - that involve increased bone resorption and a loss of bone mass. Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect the osteoclasts. The aim of this study was to obtain more information about the mechanism of action of bisphosphonates such as neridronic acid using a dual-cell culture model. As a model of osteoclastogenesis we used a murine monocyte/macrophage cell line RAW 264.7 type CRL 2278 co-cultured with murine osteoblasts. The monocyte-osteoblast system allows physiological experimentation of bone anti-resorption drugs, simulating bone turnover in pathologies such as osteoporosis. The direct actions of neridronic acid on cell proliferation and functionality in the co-culture model were examined using tartrate-resistant acid phosphatase (TRAP) assay, immunohistochemical localization of actin, and transmission and scanning electron microscopy (SEM). Results showed that the percentage of TRAP-positive cells, an early marker of osteoclastic differentiation, was significantly higher in control cultures than in co-cultures treated with variable concentrations of neridronic acid. Neridronic acid induced dramatic morphological changes, characterized by the loss of the ruffled border. The actin ring associated with the plasma membrane of the cells treated with neridronic acid was shown to break down. The tissue-specific targeting of neridronic acid to bone mineral suggests that it may inhibit bone resorption by direct effects on osteoclasts or other bone cells in the immediate microenvironment of the osteoclasts. From our study, we conclude that structural alterations induced by neridronic acid in our co-culture system lead to decreased osteoclast function. This may encourage the use of neridronic acid to reduce bone resorption in the therapy of demineralizing metabolic bone disorders.