Strategies to optimize capsid protein expression and single-stranded DNA formation of adeno-associated virus in Saccharomyces cerevisiae.

AIMS: Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that is a promising tool for gene therapy. We aimed to construct plasmids for optimal expression and assembly of capsid proteins and evaluate adenovirus (Ad) protein effect on AAV single-stranded DNA (ssDNA) formation in Saccharomyces cerevisiae. METHODS AND RESULTS: Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose-inducible promoters. Optimal VP1,2,3 expression was obtained from GAL1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus-like particles (VLPs) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ssDNA formation. CONCLUSIONS: This study confirms that yeast is able to form AAV VLPs; however, capsid assembly and ssDNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ssDNA formation. SIGNIFICANCE AND IMPACT OF THE STUDY: New manufacturing strategies for AAV-based gene therapy vectors (rAAV) are needed to reduce costs and time of production. Our study explores the feasibility of yeast as alternative system for rAAV production.

Mutations in NOTCH1 PEST domain orchestrate CCL19-driven homing of chronic lymphocytic leukemia cells by modulating the tumor suppressor gene DUSP22.

Even if NOTCH1 is commonly mutated in chronic lymphocytic leukemia (CLL), its functional impact in the disease remains unclear. Using CRISPR/Cas9-generated Mec-1 cell line models, we show that NOTCH1 regulates growth and homing of CLL cells by dictating expression levels of the tumor suppressor gene DUSP22. Specifically, NOTCH1 affects the methylation of DUSP22 promoter by modulating a nuclear complex, which tunes the activity of DNA methyltransferase 3A (DNMT3A). These effects are enhanced by PEST-domain mutations, which stabilize the molecule and prolong signaling. CLL patients with a NOTCH1-mutated clone showed low levels of DUSP22 and active chemotaxis to CCL19. Lastly, in xenograft models, NOTCH1-mutated cells displayed a unique homing behavior, localizing preferentially to the spleen and brain. These findings connect NOTCH1, DUSP22, and CCL19-driven chemotaxis within a single functional network, suggesting that modulation of the homing process may provide a relevant contribution to the unfavorable prognosis associated with NOTCH1 mutations in CLL.

CD38 in chronic lymphocytic leukemia: from bench to bedside?

Human CD38 is a cell surface molecule endowed with multiple functions. As an enzyme, it catalyzes the production of Ca2+ active metabolites, predominantly cADPR and ADPR. As a receptor, it regulates the activation of an intracellular signaling pathway, generally linked to lymphocyte activation and proliferation in physiological conditions. The finding that CD38 behaves as an independent negative prognostic factor in CLL patients was the starting point for investigations into the functional role of the molecule in the neoplastic context. Data accumulating in over a decade concur to define a model where CD38 is a central element of a large supramolecular complex that includes surface signaling receptors, chemokine receptors, adhesion molecules and matrix metalloproteases. Expression of CD38 within this supramolecular complex makes signal transduction as well as chemotaxis and homing more efficient, suggesting that the molecule is an integrator of proliferative and migratory signals. These data indicate that CD38 is not only a reliable disease marker but also a functional molecule in the CLL context. The next decade will likely tell whether it can also be a useful therapeutic target.

Unexpected differences in dissolution behavior of tablets prepared from solid dispersions with a surfactant physically mixed or incorporated.

In a previous study, it was shown that the incorporation of poorly soluble drugs (BCS class II) in sugar glasses could largely increase the drug's dissolution rate [van Drooge, D.J., Hinrichs, W.L.J., Frijlink, H.W., 2004 b. Anomalous dissolution behaviour of tablets prepared from sugar glass-based solid dispersions. J. Control. Release 97, 441-452]. However, the application of this technology had little effect when high drug loads or fast dissolving sugars were applied due to uncontrolled crystallization of the drug in the near vicinity of the dissolving tablet. To solve this problem a surfactant, sodium lauryl sulphate (SLS), was incorporated in the sugar glass or physically mixed with it. Diazepam and fenofibrate were used as model drugs in this study. The dissolution behavior of tablets prepared from solid dispersions in which SLS was incorporated was strongly improved. Surprisingly, the dissolution rate of tablets prepared from physical mixtures of SLS and the solid dispersion was initially fast, but slowed down after about 10 min. The solid dispersions were characterized by DSC to explain this unexpected difference. These measurements revealed the existence of interaction of SLS with both the drug and the sugar in the solid dispersion when SLS was incorporated. It is hypothesized that due to this interaction, the dissolution of SLS was slowed down by which a high solubility of the drug in the near vicinity of the dissolving tablet is maintained during the whole dissolution process. Therefore, uncontrolled crystallization is effectively prevented.

Radiologic progression in early rheumatoid arthritis treated with methotrexate.

OBJECTIVE: To evaluate radiologic progression in patients with early rheumatoid arthritis (RA) receiving methotrexate (MTX) as the first slow acting drug. METHODS: An open, prospective study of 29 patients with RA (21 F, 8 M, mean age 48.5+/-15.4 yrs). The mean duration of RA was 6.6 (2-60) months; and rheumatoid factor was present in 11 cases. Clinical, biological, and radiographic evaluations were done before the start of MTX treatment and after 13+/-3.8 months. Radiographs of hands and wrists were blindly studied by 2 physicians, using Larsen's and modified Sharp's methods. There was a significant correlation for the scores of the 2 physicians evaluated by kappa coefficient. Radiographic evolution was defined as an increase of 15 points in the radiologic score by each method used. RESULTS: Patients showed significant clinical improvement after one year of MTX treatment. Despite clinical and biological improvement, significant mean radiographic progression was noted, with Larsen's method (p = 0.001) and Sharp's method (p = 0.034), without reaching the maximum score. However, using the definition of radiographic progression, the radiologic scores indicated stabilization in 23 patients with Larsen's method and in 24 patients with Sharp's method. CONCLUSION: This study revealed mild radiographic progression in early RA patients treated with MTX for one year. Further controlled studies are needed.

The quality of life of people with schizophrenia in Boulder, Colorado, and Bologna, Italy.

The aim of the study was to compare the quality of life (QOL) and needs of people with schizophrenia in comprehensive treatment systems in two countries. One hundred people with schizophrenia and schizoaffective disorder were randomly selected from the caseload of a community mental health center in Boulder, Colorado, and 70 were similarly selected from public psychiatric treatment services in and around Bologna, Italy. Subjects were interviewed with QOL and needs assessment instruments and rated with the Brief Psychiatric Rating Scale. Objective QOL measures favored Bologna subjects over Boulder subjects, particularly with respect to employment, accommodation, and family life. In a factor analysis, objective QOL variables sorted separately from subjective satisfaction ratings, suggesting that they measure different underlying constructs. Patient needs in both Boulder and Bologna samples were primarily psychological and social rather than basic survival issues. Boulder subjects were more likely to report accommodation needs. Many apparent QOL advantages for Bologna subjects could be attributed to the greater frequency with which the Italian patients lived with family of origin. Living with family also appeared to confer practical benefits in meeting needs. Objective QOL measures discriminated between patient populations better than subjective ratings of satisfaction and well-being.

Effects of moderate renal insufficiency on pharmacokinetics of methotrexate in rheumatoid arthritis patients.

OBJECTIVES: To determine the effects of impaired renal function on the pharmacokinetics of methotrexate (MTX) in rheumatoid arthritis (RA) patients. METHODS: 77 RA patients were included in this study. MTX was administered intramuscularly (7.5 to 15 mg). Subjects were divided into four groups, according to their creatinine clearance (CLCR); group 1: CLCR lower than 45 ml/min; group 2: CLCR between 45 and 60 ml/min, group 3: CLCR between 61 and 80 ml/min and group 4: CLCR higher than 80 ml/min. Blood samples were collected from each subject before drug administration and at two and eight hours after administration. Individual pharmacokinetic parameters were estimated using a Bayesian approach. RESULTS: MTX concentrations (total and free) were 1.3 to 1.6-times higher in group 1 than in groups 2, 3, and 4. For total and free MTX, t1/2 eliminations were 22.7 hours in group 1, 13.5 hours in group 2, 12 hours in group 3, and 11 hours in group 4. Clearance of total MTX was 64, 92, 96, 115 ml/min in groups 1 to 4, respectively, it was 118, 163, 171, 206 ml/min in groups 1 to 4 for the free MTX, respectively. Volume of distribution averaged 2.16 l/kg in group 1, 1.92 l/kg in group 2, 1.61 l/kg in group 3, and 1.56 l/kg in group 4. Elimination half life was significantly increased and total clearance was significantly reduced with the degree of renal impairment. Linear regression revealed good correlations between clearance values of MTX and creatinine clearance. CONCLUSION: Individual testing is required rather than a general decrease of the MTX dose based only on CLCR.

Total and free methotrexate pharmacokinetics in elderly patients with rheumatoid arthritis. A comparison with young patients.

OBJECTIVE: To determine the pharmacokinetics of methotrexate (MTX) in a group of patients 65 to 83 years of age and to compare the pharmacokinetic data to those in patients 21 to 45 years of age. METHODS: Thirty-eight elderly patients (8 men, 30 women) and 24 young patients (6 men, 18 women) underwent this pharmacokinetic study. They received intramuscular administration of MTX each week, at a dose varying from 7.5 to 15 mg depending on the patient. MTX concentrations in plasma and ultrafiltrate samples were assayed by a fluorescence polarization immunoassay. Pharmacokinetic variables were estimated using a Bayesian approach with population parameters as a priori information together with 2 plasma MTX concentrations (2 and 8 h after injection). RESULTS: The extent of unbound fraction and the volume of distribution were not statistically significantly different between the 2 age groups. The elimination half-life measures of the free and total MTX were greatest in the elderly group (p < 0.001). The total clearances of free and total MTX were inversely proportional to age (p < 0.001). CONCLUSION: MTX clearance decreases with decreasing creatinine clearance and smaller doses may be chosen in this group. Thus, a dosage regimen should be adjusted in elderly patients or in those with renal impairment.

Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide.

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the proximity of the chromophore. For the dye free in solution, large spectral heterogeneity and coupling to a "bath" of low-frequency (soft) modes is observed; moreover, anharmonic motions become evident at suitably high temperatures. The average frequency of the soft modes and the amplitude of anharmonic motions depend upon solvent composition. For the DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motions are hindered. However, a new dynamic regime characterized by a large increase in anharmonic motions is observed at temperatures higher than approximately 280 K. The DNA double helix therefore appears to provide, at low temperatures, a rather rigid environment for the bound chromophore, in which conformational heterogeneity is reduced and low-frequency motions (both harmonic vibrations and anharmonic contributions) are hindered. The system becomes anharmonic at approximately 180 K; however, above approximately 280 K, anharmonicity starts to increase much more rapidly than for the dye free in solution; this can be attributed to the onset of wobbling of the dye in its intercalation site, which is likely connected with the onset of (functionally relevant) DNA motions, involving local opening/unwinding of the double helix. As shown by parallel measurements of the melting curves, these motions precede the melting of the double helix and depend upon solvent composition much more than does the melting itself.

Long-term follow-up of 453 rheumatoid arthritis patients treated with methotrexate: an open, retrospective, observational study.

A total of 453 rheumatoid arthritis (RA) patients were followed up for 35.2 +/- 27.9 months (range 3-106). The clinical parameters decreased significantly after 6 months. Twenty-eight patients were in remission (6.4%). Rheumatoid factor (RF) positivity was less common in the group of patients in remission, with a higher frequency of visits and methotrexate (MTX) onset after 65 yr. There was a significant degradation of radiographic lesions (n = 60). A total of 101 patients (23.1%) stopped MTX, for toxicity (n = 61) and failure (n = 20). The onset of MTX after 65 yr, a low number of visits and the occurrence of side-effects were predictive of MTX withdrawal. A total of 259 patients (59.3%) had side-effects. A Ritchie's index < or = 10, a lower polymorphonuclear cell count and the absence of RF were predictive of side-effects. The probability of being on MTX at 5 yr was 73%. This study confirms the high efficacy of MTX in RA.

Study of eight cases of cancer in 426 rheumatoid arthritis patients treated with methotrexate.

OBJECTIVE: To report cancer cases in 426 rheumatoid arthritis patients treated with methotrexate, and determine whether there was an increased incidence of cancer compared with patients never treated with methotrexate (rheumatoid controls) and to the whole regional population. METHODS: The duration of methotrexate treatment was 37.4 (SD 27.9) months. This population was compared with 420 rheumatoid arthritis controls and with a regional population of 812,344 people. Life table analysis was performed to compare the cancer incidence in the two rheumatoid populations. Adjustment for potentially confounding factors was done. The indirect standardisation methods was used to compare each rheumatoid population with the regional population. RESULTS: Eight cases of cancer (1.88%; 4.04 cases/1000 person years) were diagnosed in the methotrexate population v six (1.43%; 58.8 cases/1000 person years) in the rheumatoid controls. The life table method showed a higher incidence of cancer in the rheumatoid controls (P = 0.0001). In a multivariate analysis (Cox model), the only significant factor explaining this difference in the cancer incidence was age (P = 0.02). In the regional population there were 6418 new cases of cancer (0.79%; 2.85 cases/1000 person years). By the indirect standardisation method, the ratio of observed cases to expected cases of cancer in each of the rheumatoid populations was not significantly different from 1. CONCLUSIONS: In these eight cases, methotrexate was not found to be responsible for generating cancers. However, because of data regarding lymphomas and methotrexate, and because of the short follow up, especially in the control group, longer prospective studies are warranted.

Methotrexate as the initial second-line disease modifying agent in the treatment of rheumatoid arthritis patients.

OBJECTIVE: To assess the efficacy and toxicity profile of methotrexate (MTX) as the initial second-line disease modifying anti-rheumatic drug (DMARD) in rheumatoid arthritis (RA). METHODS: This was an observational retrospective cohort study comparing 28 patients who were treated with MTX as the first DMARD (MTX cases) and 55 matched patients treated with MTX after other DMARDs (MTX controls). RESULTS: The follow-up time was identical in the two groups: 19.4 +/- 14 months (2-56) for the MTX cases and 21.8 +/- 15.3 months (3-87) for MTX controls (NS). MTX efficacy was the same in the two groups, except for a higher incidence of remission in the MTX cases (8/28, 28.6% versus 5/55, 9.1%, p = 0.028). The toxicity profiles, frequencies, and reasons for MTX withdrawals were similar in the two groups. CONCLUSION: The results obtained in this study suggest a benefit from MTX prescribed as an initial second-line agent in the treatment of RA, but studies involving a larger number of patients are needed.

Mononuclear cell retention in rheumatoid synovial tissue engrafted in severe combined immunodeficient (SCID) mice is up-regulated by tumour necrosis factor-alpha (TNF-alpha) and mediated through intercellular adhesion molecule-1 (ICAM-1).

The aim of this study was to assess regulation of mononuclear cell (MNC) traffic to human synovial tissue by TNF-alpha and IL-1 and the involvement of ICAM-1 in MNC retention in rheumatoid synovial tissue. Human rheumatoid arthritis synovium was engrafted subcutaneously in 6-8 week-old SCID/CB17 mice. Three weeks later, we injected 20 x 10(6) human peripheral blood mononuclear cells (PBMC) previously labelled with 111indium intraperitoneally into mice containing control or cytokine-injected grafts. Total body scintigraphy was performed 72 h postinjection. The graft was removed and immunochemical analysis carried out to assess ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression. In some experiments, mice were treated intravenously with 500 micrograms MoAb anti-ICAM-1 (BIRR-1) or an isotype-matched control MoAb before introduction of MNC. TNF-alpha, but not IL-1 alpha, enhanced MNC retention in the rheumatoid synovial graft 72 h post-injection (graft activity 989 +/- 1227 ct/min per 200 pixels or 3.36 +/- 4.16% of initial injected activity versus 411 +/- 157 ct/min per 200 pixels or 1.13 +/- 0.45% in controls; P < 0.03). TNF-alpha enhanced ICAM-1 expression by synovial cells and endothelial cells, whereas VCAM-1 or E-selectin expression was not enhanced on either cell type. After MoAb treatment of ICAM-1, synovial lymphocyte recruitment of TNF-alpha-treated mice decreased significantly to levels below that of control mice (160 +/- 97 ct/min per 200 pixels, 0.54 +/- 0.33%; P < 0.01). Mononuclear cell retention in rheumatoid synovial tissue engrafted into SCID mice was up-regulated by TNF-alpha and blocked by MoAb to ICAM-1. These results suggest that ICAM-1 is involved in mononuclear cell retention in rheumatoid synovium.

Association of methotrexate and corticosteroids in the treatment of patients with rheumatoid arthritis.

OBJECTIVE: To investigate whether the association of methotrexate (MTX) and corticosteroids introduced concomitantly is more effective than MTX alone in patients with rheumatoid arthritis (RA). METHODS: Twenty-eight RA patients (group 1) were treated with MTX (mean dose: 10 +/- 1.4 mg/week) and corticosteroids (mean dose: 14.9 +/- 5.6 mg/day, range: 5-25) introduced concomitantly, and were compared to 251 RA patients (group 2) treated with MTX alone (mean dose: 9.8 +/-1.5 mg/week). Variations in the clinical (number of swollen and painful joints, morning stiffness, Ritchie's articular index), biological (ESR, CRP), and radiological parameters were studied. Remission was defined according to Pinals' criteria. At baseline, there were no significant differences between the two groups, except for a greater number of swollen and painful joints in group 1 (p = 0.03 and p = 0.01, respectively). The total MTX dose and the duration of treatment (26 +/- 21.8 months in group 1 versus 33.5 +/- 27.2) months in group 2) did not differ between the two groups. RESULTS: We noted a more marked reduction in the number of swollen and the number of painful joints in group 1 (p = 0.03). No differences were noted for the other clinical and biological parameters. The proportion of patients fulfilling Pinals' remission criteria was higher in group 1 (25% versus 10.1% in group 2, p = 0.04). The steroid dosage could be significantly reduced in group 1 (-3.4 +/- 6.1 mg/day, p = 0.05) and corticosteroids were stopped in 11 patients. The frequency and type of side effects, as well as the frequency and reasons leading to MTX withdrawal, did not significantly differ between the two groups. CONCLUSION: The association of MTX and corticosteroids seems to bring about a greater improvement in the different clinical activity parameters of RA than MTX alone, without any significant increase in the frequency of side effects. These results need to be confirmed in larger scale prospective studies.

Sicca syndrome associated with hepatitis C virus infection.

OBJECTIVE: To determine the prevalence of hepatitis C virus (HCV) infection in patients with sicca syndrome, and to determine the clinical, immunologic, and genetic characteristics of sicca syndrome associated with HCV. METHODS: We conducted a prospective study in a university hospital immunology-rheumatology department. Sixty-two consecutive patients with sicca syndrome according to the European criteria for Sjögren's syndrome were included. HCV infection was diagnosed in patients with positive recombinant immunoblot assay findings and the presence of viral RNA in serum and saliva. Rheumatoid factor (RF), cryoglobulins, antinuclear antibodies, and anti-SS-A/SS-B antibodies were sought. HLA typing was performed on all patients. RESULTS: The prevalence of HCV infection in patients with sicca syndrome was 19%. The incidence of neurologic involvement was significantly increased in patients with sicca syndrome associated with HCV infection (24% versus 4%; P < 0.03), as was elevations in transaminase levels (87.5% versus 16%; P < 0.0001). RF and cryoglobulins were more frequent in HCV-positive sicca syndrome patients (62% versus 30%; P < 0.03, and 56% versus 10%; P < 0.001, respectively). In contrast, anti-SS-A/SS-B antibodies were present in 38% of HCV-negative sicca syndrome patients, but in only 1 HCV-positive sicca syndrome patient (P < 0.01). No significant difference in HLA type was observed. Viral RNA was present in the saliva of 83% of HCV-positive sicca syndrome patients, but in none of the HCV-negative sicca syndrome patients. CONCLUSION: We observed a high prevalence of HCV infection in our patients with sicca syndrome. HCV-positive sicca syndrome patients had specific clinical characteristics and were seronegative for SS-A/SS-B antibodies. Moreover, HCV RNA was present in the saliva of patients with HCV-associated sicca syndrome.

[Combination regimens in rheumatoid arthritis]. / Association des traitements de fond dans la polyarthrite rhumatoïde.

The objective of combination regimens in the treatment of rheumatoid arthritis is to improve effectiveness (additive or synergistic effect) and reduce the rate of side effects. Many combinations have been tried. In general, compared with single-drug regimens, combination therapy does not increase toxicity but clinical improvement has not been proven. In addition, there is no evidence concerning the long-term effects of combination regimens. Despite these drawbacks, combining drugs known to be effective in rheumatoid arthritis alone may be an interesting alternative for patients who have responded incompletely to single drugs, particularly methotrexate. However, for both single-drug and combination regimens we are still lacking proof of a real beneficial effect in terms of reducing functional impairment, improving quality of life, lengthening life expectancy, or reducing overmortality resulting from rheumatoid arthritis. Long-term outcome may provide some answers. Finally, there are still several combinations to be studied including methotrexate with "targeted" drugs such as anti-CD4 and anti-TNF alpha antibodies or the soluble TNF alpha receptor.

IgA isotype rheumatoid factor in rheumatoid arthritis: clinical implications.

OBJECTIVES: To study the clinical, biological and radiological characteristics of RA with a predominant increase of IgA isotype rheumatoid factor (IgA-RF) over IgM-RF. METHODS: The presence of IgA-RF was determined by a sandwich-type ELISA with an antibody against the human IgA used to capture the immunoglobulin. Associated RF activity was revealed with a peroxidase-conjugated human IgG Fc fragment. Forty-nine RA patients were studied, of whom 19 had an increase in IgA-RF (38%). The control group comprised 30 RA patients without IgA-RF. RESULTS: None of the patients had isolated IgA-RF. In the selected 19 RA patients, the OD of IgA-RF (0.971 +/- 0.62 U) was higher than the IgM-RF (mean OD: 0.675 +/- 0.522 U). A statistically significant correlation was found between IgA-RF and IgM-RF (r = 0.64, p < 0.0001). No correlation was noted between the IgA concentration and the IgA-RF titer. The two groups were comparable for age, disease duration, sex ratio and previous DMARD use. We observed that patients with RA associated with increased IgA-RF more often had the sicca syndrome, but no other extra-articular features. RA patients with IgA RF also had more erosive disease: the mean Larsen score at the hand and wrist was 76 (SD = 68) versus 54 (SD = 60) in the controls, p < 0.02. Replacement surgery for the hip or knee was necessary in 47% of the RA patients with IgA-RF, versus 13% in the controls, p < 0.01. No association of IgA-RF with disease activity was noted. CONCLUSION: Our study showed that RA patients with a predominant increase of IgA-RF had a more erosive disease and a high frequency of associated sicca syndrome.

Effect of age on the efficacy and tolerance of methotrexate in rheumatoid arthritis.

The objective of this study was to assess the influence of age on the efficacy and toxicity of methotrexate in rheumatoid arthritis. Four hundred and sixty-nine patients were separated according to the age of onset of methotrexate treatment: before 65 yr (group 1, n = 416) and after 65 yr (group 2, n = 53). No difference was found in the evolution of clinical and biological parameters between the two groups. The number of patients in remission at the end of the study was equal. The frequency and type of side-effects were similar. No significant difference was found in the frequency and reasons for methotrexate withdrawal. We noted a trend towards lower therapeutic maintenance of methotrexate when prescribed after the age of 65 yr (P = 0.07, actuarial method). In conclusion, the age at initiation of methotrexate treatment probably did not influence its efficacy and toxicity in rheumatoid arthritis.