[Show abstract][Hide abstract] ABSTRACT: Objective
We previously reported that selective depletion of B-lymphocytes with rituximab, an anti-CD20 monoclonal antibody, slowed decline of beta-cell function in recent-onset type 1 diabetes mellitus (T1DM) at one year. Subjects were followed further to determine whether there was persistence of effect.Research Design and Methods
Eighty-seven subjects (ages 8-40) were randomly assigned to, and 81 received, infusions of rituximab or placebo on days 1, 8, 15, and 22. The primary outcome - baseline-adjusted mean 2-hour area under the curve (AUC) serum C-peptide during a mixed meal tolerance test (MMTT) at one year - showed higher C-peptide AUC with rituximab versus placebo. Subjects were further followed with additional MMTTs every 6 months.ResultsThe rate of decline of C-peptide was parallel between groups, but shifted by 8.2 months in rituximab treated subjects. Over 30 months, AUC, insulin dose, and HbA1c were similar for rituximab and placebo. However, in evaluating change in C-peptide over the entire follow-up period, the rituximab group means were significantly larger as compared within assessment times to the placebo group means using a global test (p = 0.03). Odds ratio for loss of C-peptide to < 0.2 nmol/L following rituximab was 0.565 (p= 0.064). B-lymphocytes recovered to baseline values by 18 months. Serum IgG levels were maintained in the normal range but IgM levels were depressed.Conclusions
Like several other immunotherapeutic approaches tested, in recent-onset T1DM, rituximab delays the fall in C-peptide, but does not appear to fundamentally alter the underlying pathophysiology of the disease.
[Show abstract][Hide abstract] ABSTRACT: Bacterial adherence to the acquired dental pellicle, important in dental caries (caries), is mediated by receptor-adhesins such as salivary agglutinin binding to Streptococcus mutans antigen I/II (I/II). Ten selected I/II epitopes were chosen to determine their reactivity to human salivary IgA. Previous studies suggested that a specific HLA biomarker group (HLA-DRB1*04) may have differential influence of immune responses to I/II. However, it was not known whether secretory IgA (SIgA) responses to the selected epitopes from HLA-DRB1*04 positive subjects were different compared to controls, or across other caries-related factors such as total IgA (TIgA). Thirty-two total subjects were matched accord-ing to HLA type, gender, ethnicity and age. HLA genotyping, oral bacterial, immunoglobulin and antibody analyses were performed. A large observed difference emerged with regard to the natural immune reservoir of TIgA in HLA-DRB1*04 positive subjects, specifically, a 27.6% reduction compared to controls. In contrast to all other epitopes studied, HLA-DRB1*04 positive sub-jects also exhibited reduced reactivity to I/II epitope 834-853. HLA-DRB1*04 positive subjects exhibited lower specific SIgA activity/TIgA to 834-853 and also a lower specific reactivity to 834-853/whole cell S. mutans UA159. Further-more, HLA-DRB1*04 positive subjects exhibited lower responses to I/II in its entirety. The large observed difference in TIgA and the 834-853 re-activity pattern across multiple measures suggest potentially important connections pertaining to the link between HLA-DRB1*04 and caries.
Open Journal of Immunology 09/2013; 3(3):82-92.
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[Show abstract][Hide abstract] ABSTRACT: The upper age of renal transplant recipients is rising on the transplant wait list. Age-dependent immune responsiveness to new antigens has not been thoroughly studied. This study used a mouse model of alloantibody response to neoalloantigen to study age-related differences.
Transgenic huCD20-C57BL/6 mice were immunized intraperitoneally with BALB/c splenocytes (2.5 × 10(7)) at baseline and 1 month. Plasma samples were collected at baseline and 1 and 2 months after inoculation, frozen, and tested in a batch run (n = 22). Samples were tested by flow cytometric crossmatch for alloantibody with 2-fold serial dilution from neat to 1:640 using BALB/c splenocytes as targets. The sum of the median fluorescence intensity of the tested sample was calculated after subtracting that of an autologous serum control. Elderly mice (ELD; 42-103 weeks) at inoculation were compared with younger mice (YOU; 11-15 weeks). Statistical analysis was performed with 2-sample t test.
Mean age (weeks) between the groups was significantly different (ELD 69.3 ± 9.6 vs YOU 13.4 ± 1.4; P < .001). There was no difference in alloantibody between groups at baseline (ELD 0.7 ± 3.1 vs YOU 0.6 ± 0.4; P = .93). There was a higher alloantibody response at 1 month for YOU (52.9 ± 31.78) compared with ELD (5.12 ± 8.18). There was a greater difference after the 2 month (YOU 109.38 ± 66.43 vs ELD 21.97 ± 27.14; P < .0024).
There was a difference in response to new alloantigen in this animal model. Older animals had significantly decreased responses to new alloantigen stimulation 1 month after inoculation and even more profound decreases at 2 months compared with young animals. This model may be used to study differences in immune refractoriness to antigen signaling. It may be important to adapt clinical immunosuppression in the aged population to possible decreased responses to immune stimulation.
[Show abstract][Hide abstract] ABSTRACT: While several studies have examined the general inflammatory responses in relation to cytomegalovirus infection, the identification of the various inflammatory mediators as well as their relative importance is far from clear.
Solid organ recipients enrolled in an international multicenter trial of cytomegalovirus disease treatment (the VICTOR study) were analyzed (n = 289) (ClinicalTrials.gov NCT00431353). Plasma markers of inflammation and endothelial cell activation were assessed at baseline by enzyme immunoassays.
THE MAJOR FINDINGS WERE: (i) Plasma levels of the CXC-chemokine interferon-inducible protein-10 (P<0.001) and C-reactive protein (P = 0.046) were independently associated with the presence of cytomegalovirus DNAemia above lower level of quantification. (ii) High levels of CC-chemokine ligand 21 (P = 0.027) and pentraxin 3 (P = 0.033) were independently associated with tissue invasive cytomegalovirus disease as opposed to cytomegalovirus syndrome.
Our findings illustrate the complex interaction between cytomegalovirus and the immune system, involving a wide range of inflammatory mediators that could be associated to disease manifestations in cytomegalovirus related disease.
PLoS ONE 04/2013; 8(4):e60767. · 3.53 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Rituximab has been successfully used as an experimental therapy in different autoimmune diseases. Recently, a double-blind placebo-controlled phase-2 study in early onset type 1 diabetes showed that rituximab delayed progression of the disease. However, like with any immunosuppressive therapy, there is a concern of opportunistic viral reactivations with the use of rituximab, including herpes and polyomaviruses. OBJECTIVES: To study the incidence of new infections and reactivations with BK, JC, Epstein-Barr and cytomegalovirus (BKV, JCV, EBV and CMV) in T1D participants in the phase-2 rituximab study. STUDY DESIGN: Subjects received 4 weekly doses of rituximab (N=57) or placebo (N=30) during the first month of study. Blood samples obtained at weeks 0, 12, 26, 56 and 78 were assayed for CMV, EBV, BKV and JCV by real-time DNA PCR and serology. RESULTS: EBV reactivations were diagnosed by PCR in 25% of placebo, but none of rituximab recipients (p<0.01). There were no episodes of CMV viremia in either treatment group. BKV viremias were significantly more common in the rituximab recipients (9%) compared with placebo controls (0, p<0.01). No JCV reactivations were detected in this study, but among 6 rituximab and 2 placebo recipients who seroconverted for JCV during the study, only one rituximab recipient had detectable viremia. All infections were asymptomatic. CONCLUSIONS: Four doses of rituximab administered to individuals with early onset T1D decreased the incidence of asymptomatic EBV reactivations, as predicted by the rituximab-mediated elimination of memory B-cells, but increased the frequency of asymptomatic viremias caused by polyomaviruses.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 02/2013; · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyomavirus BK (BKV)-associated nephropathy causes premature kidney transplant (KT) failure. BKV viruria and viremia are biomarkers of disease progression, but associated risk factors are controversial. A total of 682 KT patients receiving basiliximab, mycophenolic acid (MPA), corticosteroids were randomized 1:1 to cyclosporine (CsA) or tacrolimus (Tac). Risk factors were analyzed in 629 (92.2%) patients having at least 2 BKV measurements until month 12 posttransplant. Univariate analysis associated CsA-MPA with lower rates of viremia than Tac-MPA at month 6 (10.6% vs. 16.3%, p = 0.048) and 12 (4.8% vs. 12.1%, p = 0.004) and lower plasma BKV loads at month 12 (3.9 vs. 5.1 log(10) copies/mL; p = 0.028). In multivariate models, CsA-MPA remained associated with less viremia than Tac-MPA at month 6 (OR 0.60; 95% CI 0.36-0.99) and month 12 (OR 0.33; 95% CI 0.16-0.68). Viremia at month 6 was also independently associated with higher steroid exposure until month 3 (OR 1.19 per 1 g), and with male gender (OR 2.49) and recipient age (OR 1.14 per 10 years) at month 12. The data suggest a dynamic risk factor evolution of BKV viremia consisting of higher corticosteroids until month 3, Tac-MPA compared to CsA-MPA at month 6 and Tac-MPA, older age, male gender at month 12 posttransplant.
American Journal of Transplantation 11/2012; · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Treatment failure or relapse is common in solid organ transplant recipients treated for cytomegalovirus (CMV) disease. Because CMV infections induce a vigorous inflammatory response, we investigated whether pretreatment levels of inflammatory markers were associated with virologic and clinical outcomes. PATIENTS AND METHODS: Solid organ transplant recipients enrolled in an international multicenter trial of CMV disease treatment (the VICTOR study) were studied (n=248). Plasma levels of markers of inflammation and endothelial cell activation were assessed at baseline and during follow-up by enzyme immunoassays. RESULTS: Baseline values for the chemokine CXCL16 was an independent predictor of clinical outcome (P=0.003) and was a weak independent predictor of suppression of viral load below level of detection (LOD) (P=0.013) at day 21 after initiation of treatment. Baseline levels of the long pentraxin 3 (PTX3) was an independent predictor of suppression of viral load below LOD at day 21 (P=0.002), whereas baseline levels of von Willebrand factor (vWF) was an independent predictor of clinical outcome at day 21 (P=0.008), and vWF levels at day 21 was a weak independent inflammatory predictor of viral recurrence (P=0.018). CONCLUSION: The present study shows that the plasma levels of CXCL16, PTX3 and vWF at the start of treatment are independently associated with virologic and clinical treatment failure during anti-CMV therapy in solid organ transplant recipients. These findings suggest a link between CMV infection and inflammation that also may influence the outcome of anti-CMV therapy.
[Show abstract][Hide abstract] ABSTRACT: Interpretation of clinical trials to alter the decline in β-cell function after diagnosis of type 1 diabetes depends on a robust understanding of the natural history of disease. Combining data from the Type 1 Diabetes TrialNet studies, we describe the natural history of β-cell function from shortly after diagnosis through 2 years post study randomization, assess the degree of variability between patients, and investigate factors that may be related to C-peptide preservation or loss. We found that 93% of individuals have detectable C-peptide 2 years from diagnosis. In 11% of subjects, there was no significant fall from baseline by 2 years. There was a biphasic decline in C-peptide; the C-peptide slope was -0.0245 pmol/mL/month (95% CI -0.0271 to -0.0215) through the first 12 months and -0.0079 (-0.0113 to -0.0050) from 12 to 24 months (P < 0.001). This pattern of fall in C-peptide over time has implications for understanding trial results in which effects of therapy are most pronounced early and raises the possibility that there are time-dependent differences in pathophysiology. The robust data on the C-peptide obtained under clinical trial conditions should be used in planning and interpretation of clinical trials.
[Show abstract][Hide abstract] ABSTRACT: The impact of different cytomegalovirus (HCMV) glycoprotein B (gB) genotypes on pathogenesis remains controversial.
To investigate the effect of gB genotypes either as single infections or as part of multiple infections on the early kinetics of response to ganciclovir therapy.
Patients (n=239) enrolled in a study of intravenous ganciclovir or valganciclovir for the treatment of HCMV disease were analysed by a gB genotype specific PCR to quantify the amount of each gB genotype present at initiation of therapy (baseline, day 0) and at days 3, 7, 14 and 21 post therapy.
In all gB groups (individual gB genotype infections and mixed genotype infections) there was a biphasic decline in viral load after therapy. The first phase half life (days 0-3) was ≤1 day and was followed over the next 18 days by a slower second phase decline with half lives ranging from 3.4 to 4.4 days. The 1st phase rapid decline in viral load was dependent upon gB genotype whereas the ultimate viral load reduction at day 21 was relatively insensitive to gB genotype. A strong correlation between 1st phase decline and extent of viral load reduction at day 21 was observed (r=0.37; p=0.002). These data imply that early reductions in HCMV load after therapy may be useful in predicting the duration of drug therapy needed to control HCMV replication.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 03/2012; 54(1):56-60. · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This prospective pharmacoepidemiological study examined treatment and outcomes in patients converted to sirolimus (SRL) after renal transplantation. 484 subjects in 36 centres in 7 countries were followed for up to 5 years. Principal reasons for conversion were declining graft function (146/484, 30%) and side effects of prior therapy (144/484, 30%) and the major treatment combinations after conversion were SRL ± MMF (62%), SRL + TAC (21.5%), SRL + CSA (16.5%). The cumulative probability of biopsy-confirmed acute rejection (BCAR) was 5% (n = 22), death-censored graft loss 12% (n = 56) and death 6% (n = 22), and there was no significant relationship to the treatment combination employed. Median calculated creatinine clearance was 48.4 (29.3, 64.5) mL/min at conversion, rising to 54.1 (41.2, 69.0) mL/min at month 1, 55.7 (39.0, 73.0) mL/min at month 12, 58.6 (39.7, 75.2) mL/min at two years and 60.9 (36.0, 77.0) mL/min at three years post-conversion. The most common adverse events were hypertension (47%), hyperlipidemia (26%), urinary tract infections (25%), anaemia (24%) and diarrhea (14%), and cardiac events, hyperlipemia and CMV infection were more common in patients converted during the first year. SRL was most frequently combined with MMF after conversion, but principal clinical outcomes were not significantly influenced by the treatment combination employed in normal practice.
[Show abstract][Hide abstract] ABSTRACT: Preservation of β-cell function as measured by stimulated C-peptide has recently been accepted as a therapeutic target for subjects with newly diagnosed type 1 diabetes. In recently completed studies conducted by the Type 1 Diabetes Trial Network (TrialNet), repeated 2-hour Mixed Meal Tolerance Tests (MMTT) were obtained for up to 24 months from 156 subjects with up to 3 months duration of type 1 diabetes at the time of study enrollment. These data provide the information needed to more accurately determine the sample size needed for future studies of the effects of new agents on the 2-hour area under the curve (AUC) of the C-peptide values. The natural log(x), log(x+1) and square-root (√x) transformations of the AUC were assessed. In general, a transformation of the data is needed to better satisfy the normality assumptions for commonly used statistical tests. Statistical analysis of the raw and transformed data are provided to estimate the mean levels over time and the residual variation in untreated subjects that allow sample size calculations for future studies at either 12 or 24 months of follow-up and among children 8-12 years of age, adolescents (13-17 years) and adults (18+ years). The sample size needed to detect a given relative (percentage) difference with treatment versus control is greater at 24 months than at 12 months of follow-up, and differs among age categories. Owing to greater residual variation among those 13-17 years of age, a larger sample size is required for this age group. Methods are also described for assessment of sample size for mixtures of subjects among the age categories. Statistical expressions are presented for the presentation of analyses of log(x+1) and √x transformed values in terms of the original units of measurement (pmol/ml). Analyses using different transformations are described for the TrialNet study of masked anti-CD20 (rituximab) versus masked placebo. These results provide the information needed to accurately evaluate the sample size for studies of new agents to preserve C-peptide levels in newly diagnosed type 1 diabetes.
PLoS ONE 11/2011; 6(11):e26471. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytomegalovirus (CMV) displays genetic polymorphisms in multiple genes, which may result in important virulence differences. Glycoprotein N (gN) and immediate early 1 (IE1) are key viral genes and immune targets. We aimed to characterize the molecular epidemiology of gN and IE1 genotypes in organ transplant patients with CMV disease in the context of clinical and virologic endpoints.
A total of 240 patients with CMV disease had genotyping analysis by nested polymerase chain reaction assays and sequencing using blood samples obtained at disease onset. Results were correlated with viral clearance kinetics and recurrence.
Complex patterns of gN and IE1 genotypes were present with no clear genetic linkages. No single genotype of IE1 or gN was associated with poorer outcome. For example, different gN or IE1 genotypes had comparable baseline viral load, clearance half-lives, time to clearance, and rates of virologic recurrence. Mixed infection was present at IE1 in 15.8% and gN in 21.9%, but analysis of a single gene was insufficient to detect all mixed infections. Infections caused by multiple strains, as opposed to single strains, were associated with higher baseline viral loads (P = 0.011), delayed viral clearance (P = 0.033), and higher rates of virologic recurrence (P = 0.008).
Genetic diversity in CMV is complex. Specific gN or IE subtypes do not seem to affect in vivo viral virulence patterns in single-strain infections. Mixed infections demonstrate associations with virologic outcomes that single-strain infections do not.
[Show abstract][Hide abstract] ABSTRACT: B-lymphocyte depletion with rituximab has been shown to benefit patients with various autoimmune diseases. We have previously demonstrated that this benefit is also apparent in patients with newly diagnosed type 1 diabetes.
The effect of rituximab on in vivo antibody responses, particularly during the period of B-lymphocyte depletion, is incompletely determined. This study was designed to assess this knowledge void.
In patients with recent-onset type 1 diabetes treated with rituximab (n = 46) or placebo (n = 29), antibody responses to neoantigen phiX174 during B-lymphocyte depletion and with hepatitis A (as a second neoantigen) and tetanus/diphtheria (as recall antigens) after B-lymphocyte recovery were studied. Anti- tetanus, diphtheria, mumps, measles, and rubella titers were measured before and after treatment by means of ELISA. Antibody titers and percentage IgM versus percentage IgG to phiX174 were measured by means of phage neutralization. B-lymphocyte subsets were determined by means of flow cytometry.
No change occurred in preexisting antibody titers. Tetanus/diphtheria and hepatitis A immunization responses were protective in the rituximab-treated subjects, although significantly blunted compared with those seen in the controls subjects, when immunized at the time of B-lymphocyte recovery. Anti-phiX174 responses were severely reduced during the period of B-lymphocyte depletion, but with B-lymphocyte recovery, anti-phiX174 responses were within the normal range.
During the time of B-lymphocyte depletion, rituximab recipients had a decreased antibody response to neoantigens and significantly lower titers after recall immunization with diphtheria and tetanus toxoid. With recovery, immune responses return toward normal. Immunization during the time of B-lymphocyte depletion, although ineffective, does not preclude a subsequent response to the antigen.
The Journal of allergy and clinical immunology 09/2011; 128(6):1295-1302.e5. · 12.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type 1 diabetes mellitus is believed to be due to the autoimmune destruction of β-cells by T lymphocytes, but a single course of rituximab, a monoclonal anti-CD20 B lymphocyte Ab, can attenuate C-peptide loss over the first year of disease. The effects of B cell depletion on disease-associated T cell responses have not been studied. We compare changes in lymphocyte subsets, T cell proliferative responses to disease-associated target Ags, and C-peptide levels of participants who did (responders) or did not (nonresponders) show signs of β-cell preservation 1 y after rituximab therapy in a placebo-controlled TrialNet trial. Rituximab decreased B lymphocyte levels after four weekly doses of mAb. T cell proliferative responses to diabetes-associated Ags were present at baseline in 75% of anti-CD20- and 82% of placebo-treated subjects and were not different over time. However, in rituximab-treated subjects with significant C-peptide preservation at 6 mo (58%), the proliferative responses to diabetes-associated total (p = 0.032), islet-specific (p = 0.048), and neuronal autoantigens (p = 0.005) increased over the 12-mo observation period. This relationship was not seen in placebo-treated patients. We conclude that in patients with type 1 diabetes mellitus, anti-B cell mAb causes increased proliferative responses to diabetes Ags and attenuated β-cell loss. The way in which these responses affect the disease course remains unknown.
The Journal of Immunology 08/2011; 187(4):1998-2005. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In patients with cytomegalovirus (CMV) disease, regular monitoring of viral loads and treatment until negative are recommended. However, with more sensitive polymerase chain reaction (PCR) assays and cellular peripheral sample types, detection of low-level viremia is achievable. We compared a whole blood real-time PCR with a plasma PCR assay for monitoring therapeutic response.
Patients enrolled in a trial to treat CMV disease for 21 days had regular viral load monitoring. The results of a plasma-based PCR assay were compared with a real-time PCR assay of whole blood and assessed for their ability to predict recurrence.
In 219 evaluable patients, viral loads in plasma versus whole blood demonstrated good correlation but significant difference in absolute value and clearance kinetics. Virus was still detectable by day 21 in 154 of 219 (70.3%) patients with the whole blood versus 105 of 219 (52.1%; P<0.001) patients with the plasma assay. The positive predictive value of persistent plasma viremia at day 21 for virologic recurrence was 41.9% vs. 36.3% for the whole blood assay. In the subset of patients with a negative plasma but positive whole blood at day 21 (n = 49), the incidence of virologic recurrence was similar to that of all patients with a negative plasma assay (23.1% vs. 23.6%).
When treating CMV disease, enhanced detection of residual viremia using a whole blood real-time PCR does not seem to offer significant clinical advantages nor allows for better prediction of recurrence of CMV viremia or disease. The treat-to-negative paradigm may not hold true when such assays are used.
[Show abstract][Hide abstract] ABSTRACT: An effective host immune response, critical for successful control of Cytomegalovirus (CMV) disease in solid organ transplant recipients, is affected by intensity and type of immunosuppressive therapy. We used information prospectively captured in the VICTOR-trial to investigate the impact of immunosuppressive therapy on short- and long-term outcomes of CMV treatment in organ transplant recipients. Dual, as compared to triple, immunosuppressive therapy ([odds ratios] OR of 2.55; 95% CI: 1.51-4.60; p = 0.002), lower blood concentrations of calcineurin inhibitors (OR of 5.53; CI: 1.04-29.35; p = 0.045), and longer time since transplantation (OR of 1.70; CI: 1.01-2.87; p = 0.047) all showed better early (Day 21) CMV DNAemia eradication. We observed no effect of the intensity of the immunosuppressive therapy on overall rates of viral eradication or recurrence. The type of calcineurin inhibitor (tacrolimus/cyclosporine) or use of mycophenolate did not affect treatment efficacy, although both tacrolimus and mycophenolate treated patients showed a lower rate of virological recurrence OR 0.51 (95% CI: 0.26-0.98; p = 0.044) and OR 0.45 (95% CI: 0.22-0.93; p = 0.031), respectively. Lower total intensity of immunosuppressive therapy was associated with more effective early, but not overall, CMV DNAemia eradication by valganciclovir/ganciclovir therapy. Both mycophenolate and tacrolimus (rather than cyclosporine) therapy seem to be associated with reduced risk of recurrence.
American Journal of Transplantation 08/2010; 10(8):1881-8. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In 2001 valganciclovir was approved by the FDA for treatment of HIV associated retinitis and in 2003 for prevention of post transplant CMV. This review provides an update on the status of its use and areas of controversy: How long should prophylaxis be given?; What is the appropriate dose for prophylaxis?; Can it be used in children, and at what dose?; Can it be used to treat CMV disease? The question of optimal dosing will probably not be settled as the sample size for controlled trials would be prohibitive. Other trials clearly show that extended therapy provides added benefit, the drug is safe and an appropriate dose has been identified in children and oral therapy of CMV disease is effective.
American Journal of Transplantation 06/2010; 10(6):1359-64. · 6.19 Impact Factor