Nilotinib Treatment of Patients Affected by Chronic Graft-versus-Host Disease Reduces Collagen Production and Skin Fibrosis by Downmodulating the TGF-ß and p-SMAD Pathway.

The present study was conducted to investigate cellular and molecular features of chronic graft-versus-host disease fibroblasts (GVHD-Fbs) and to assess the effectiveness of nilotinib as a fibrosis modulator. Growth kinetics, phenotype, and differentiation of cultured skin biopsy-derived GVHD-Fbs were compared with normal fibroblasts from both a dermal cell line (n-Fbs) and healthy individuals undergoing cosmetic surgery (n-skin-Fbs). Collagen genes (COL1α1/COL1α2) and p-SMAD2 expression were assessed by real-time PCR and immunofluorescence. The in vivo effects of nilotinib on chronic GVHD (cGVHD)-affected skin were investigated by immunohistochemistry; the relationship to TGF-ß plasma levels was assessed. Although the morphology, phenotype, and differentiation of cultured GVHD-Fbs were comparable to normal fibroblasts, growth was slower and senescence was reached earlier. The expression of COL1α1 and COL1α2 mRNAs was respectively 4 and 1.6 times higher in cGVHD-Fbs (P = .02); the addition of TGF-ß increased n-Fbs, but not GVHD-Fbs, collagen gene expression. Compared with the baseline, the addition of 1 µM nilotinib induced 86.5% and 49% reduction in COL1α1 and COL1α2 expression in cultured GVHD-Fbs, respectively (P< .01). In vivo immunohistochemistry analysis of skin biopsy specimens from patients with cGVHD showed strong baseline staining for COL1α1 and COL1α2, which decreased sharply after 180 days of nilotinib; immunofluorescence revealed TGF-ß inhibition and p-Smad2 reduction at the intracellular level. Of note, nilotinib treatment was associated with normalization of TGF-ß levels both in culture supernatants and in plasma. In general, the data show that cGVHD fibroblasts promote fibrosis through abnormal collagen production induced by hyperactive TGF-ß signaling. TGF-ß inhibition at the intracellular and systemic level represents an essential antifibrotic mechanism of nilotinib in a clinical setting.

A novel method to evaluate prethawing viability of cryopreserved CD34+ hematopoietic stem cells for autologous transplantation.

BACKGROUND: Cryopreservation of CD34+ hematopoietic stem cells (HSCs) is associated with variable loss of viability. Although postfreezing CD34+ cell viability can be assessed on the sampling tube (bag tail) directly connected to the main bag (mother bag), results often underestimate the actual viability observed when the mother bag is thawed and reinfused. We assessed a novel method to measure postfreezing CD34+ cell viability, based on small bag (minibag) samples; results were compared with those obtained on the corresponding mother bags and bag tails. STUDY DESIGN AND METHODS: Sixty-one apheresis procedures of 42 patients undergoing autologous HSC transplant were analyzed. Viable CD34+ cells were quantified with flow cytometry before controlled rate freezing (ICE-CUBE14M system, SY-LAB- IceCube, SIAD), after 10 days of storage (mini-bag and bag tail), and before reinfusion (aliquot from a thawed mother bag). Results were compared using Student's t test and Spearman's rho correlation test. RESULTS: The mean CD34+ cell viability before cryopreservation was 99.3% (confidence interval [CI], 98.94-99.65%); the mean amount of CD34+ cells, white blood cells and neutrophils in the mother bag was 0.8 ± 1.1 × 109 /L, 63.4 ± 23.5 × 109 /L, and 25.7 ± 15.5 × 109 /L, respectively. Mother bags postthawing CD34+ cell viability was 72.3% (CI, 67.74-76.85%; p < 0.01 compared to prefreezing); no difference was observed with respect to minibags (73.7%; CI, 69.80-77.59%; p = NS), whereas significantly lower values were found for bag tails (58.6%; CI, 54.19-63.00%; p < 0.01 vs. both mini- and mother bags). CONCLUSION: Compared to bag tails, minibags represent a more accurate tool to measure the CD34+ cell viability of the apheresis mother bag prior to reinfusion; in addition, minibags may could be of help for case-by-case calculation of the amount of apheresis to be infused to patients undergoing autologous HSC transplant.

Immunomodulatory Effects of Tyrosine Kinase Inhibitor In Vitro and In Vivo Study.

Pathogenesis of chronic graft-versus-host disease (cGVHD) is incompletely defined, involving donor-derived CD4 and CD8-positive T lymphocytes as well as B cells. Standard treatment is lacking for steroid-dependent/refractory cases; therefore, the potential usefulness of tyrosine kinase inhibitors (TKIs) has been suggested, based on their potent antifibrotic effect. However, TKIs seem to have pleiotropic activity. We sought to evaluate the in vitro and in vivo impact of different TKIs on lymphocyte phenotype and function. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in the presence of increasing concentrations of nilotinib, imatinib, dasatinib, and ponatinib; in parallel, 44 PBMC samples from 15 patients with steroid-dependent/refractory cGVHD treated with nilotinib in the setting of a phase I/II trial were analyzed at baseline, after 90, and after 180 days of therapy. Flow cytometry was performed after labeling lymphocytes with a panel of monoclonal antibodies (CD3, CD4, CD16, CD56, CD25, CD19, CD45RA, FoxP3, CD127, and 7-amino actinomycin D). Cytokine production was assessed in supernatants of purified CD3+ T cells and in plasma samples from nilotinib-treated patients. Main T lymphocyte subpopulations were not significantly affected by therapeutic concentrations of TKIs in vitro, whereas proinflammatory cytokine (in particular, IL-2, IFN-γ, tumor necrosis factor-α, and IL-10) and IL-17 production showed a sharp decline. Frequency of T regulatory, B, and natural killer (NK) cells decreased progressively in presence of therapeutic concentrations of all TKIs tested in vitro, except for nilotinib, which showed little effect on these subsets. Of note, naive T regulatory cell (Treg) subset accumulated after exposure to TKIs. Results obtained in vivo on nilotinib-treated patients were largely comparable, both on lymphocyte subset kinetics and on cytokine production by CD3-positive cells. This study underlines the anti-inflammatory and immunomodulatory effects of TKIs and supports their potential usefulness as treatment for patients with steroid-dependent/refractory cGVHD. In addition, both in vitro and in vivo data point out that compared with other TKIs, nilotinib could better preserve the integrity of some important regulatory subsets, such as Treg and NK cells.

Molecular analysis of hepatitis B virus (HBV) in an HIV co-infected patient with reactivation of occult HBV infection following discontinuation of lamivudine-including antiretroviral therapy.

BACKGROUND: Occult hepatitis B virus (HBV) infection (OBI) is characterized by HBV DNA persistence even though the pattern of serological markers indicates an otherwise resolved HBV infection. Although OBI is usually clinically silent, immunocompromised patients may experience reactivation of the liver disease. CASE PRESENTATION: We report the case of an individual with human immunodeficiency virus (HIV) infection and anti-HBV core antibody positivity, who experienced severe HBV reactivation after discontinuation of lamivudine-including antiretroviral therapy (ART). HBV sequencing analysis showed a hepatitis B surface antigen escape mutant whose presence in an earlier sample excluded reinfection. Molecular sequencing showed some differences between two isolates collected at a 9-year interval, indicating HBV evolution. Resumption of ART containing an emtricitabine/tenofovir combination allowed control of plasma HBV DNA, which fell to undetectable levels. CONCLUSION: This case stresses the ability of HBV to evolve continuously, even during occult infection, and the effectiveness of ART in controlling OBI reactivation in HIV-infected individuals.

The effect of dupilumab in an HBV-HIV coinfected atopic patient: a case report.

Atopic dermatitis (AD) is a chronic immune-mediated inflammatory disease typical of childhood that can also affect adults. AD is clinically characterized by intensely pruritic eczematous lesions. The burden of this disease and its impact on quality of life are often substantial. Dupilumab is a fully humanized monoclonal antibody against interleukin 4 (IL-4) receptor α, capable of blocking IL-4 and IL-13 signaling. This novel therapy represents the first biologic approved for the treatment of moderate to severe AD. Our report describes the case of a 39-year-old adult patient affected by severe chronic AD with associated allergic and viral comorbidities for whom conventional systemic therapies proved ineffective or contraindicated. The main source of interest in this case is hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection because, to our knowledge, this is the first case of an adult atopic patient treated with dupilumab in the simultaneous presence of these comorbidities. Regarding coinfections, the patient was on antiretroviral therapy for HBV and HIV before starting dupilumab. Efficacy and safety data after 24 weeks of therapy are reported in detail.

[Durability and tolerability of long-term nevirapine-based HAART]. / Tollerabilità metabolica e durevolezza degli schemi antiretrovirali comprendenti nevirapina.

Efficacy and tolerability of nevirapine-based HAART regimens were retrospectively evaluated. HIV-1-infected patients were included if they had been receiving a NVP-containing HAART regimen for at least 60 months, regardless of the reason for its initiation. A total of 82 patients were included in this study. The median follow-up time period was 96 months (range 64-120). At the time of starting NVP-based therapy, 31.7% (26 patients) were antiretroviral-naive, while 68.3% (56 patients) switched to an NVP-based strategy because of intolerance to the prior regimen, failure of a previous regimen or for simplification purposes. Coinfection with hepatitis B or C viruses was present in 26.8% of the patients (n 22). The most frequent nucleoside analogue backbone was zidovudine/lamivudine (52.4%) followed by stavudine/lamivudine (15.8%). A protease inhibitor was concomitantly administered in 2.4% of cases. To investigate the tolerability, we report the results separately on the basis of the presence/absence of HBV and/or HCV coinfection. Coinfected patients displayed significantly higher baseline liver enzymes than non-coinfected patients. Nevertheless, ALT levels showed a constant decrease over time in coinfected patients. Overall, median triglycerides, HDL and total cholesterol values tended to be lower in coinfected patients than in the remaining subjects. In particular, there was a significant decrease in triglyceride values in both groups and, concomitantly, a slight increase in total cholesterol. On the other hand, HDL cholesterol demonstrated a progressive increase. Finally, no change was found in glucose plasma levels, which always remained within normal ranges. In conclusion, no long-term toxicities associated with the use of nevirapine beyond five years were documented in our cohort of patients, even in coinfected patients. Long-term exposure to nevirapine was associated with optimal HIV suppression and favourable lipidic and glucidic profiles.

Day-On, Day-Off emtricitabine, tenofovir disoproxil fumarate and efavirenz single tablet regimen (DODO) as maintenance therapy in HIV-infected patients.

Reduced dose schedules may be feasible options to simplify antiretroviral therapy (ART) in selected HIV-1-infected individuals. Efficacy and safety of a Day-on, Day-off (DODO) schedule of tenofovir disoproxil fumarate, emtricitabine and efavirenz (FTC/TDF/EFV) single tablet regimen (STR) was assessed. Twenty-seven patients were prescribed the DODO schedule and were monitored for 48 weeks. Switching criteria were: no previous ART failure, no AIDS-defining illnesses, T CD4 cell nadir >200/mmc, and HIV-RNA below detection limit (40 copies/mL) for at least six months. Clinical and laboratory data, including plasma HIV-RNA levels, T CD4 and CD8 counts, liver and kidney function, lipid levels and ultrasensitive C-reactive protein (us-CRP) were assessed at baseline, week 4, 12, 24, and 48. Statistical analysis was performed by paired Student's T-test for comparison between baseline and each time point and Chi square test for CD4/CD8 ratio comparison. In all, 26 out of 27 patients maintained plasma HIV-RNA levels below the detection limit through the entire follow-up. One patient experienced low level plasma HIV-RNA rebound at week 36 (47 copies/ml) and immediately reverted to the conventional dose schedule of FTC/TDF/EFV; plasma HIV-RNA was undetectable after four weeks. No major changes on liver and kidney function tests, lipid levels and us-CRP were observed. Although no profound modifications of T CD4 count were observed during follow-up, the CD4/CD8 ratio increased significantly at week 48 compared to the baseline (p<0.05). In conclusion, 48-week DODO administration of the fixed dose FTC/TDF/EFV STR combination was safe and effective in maintaining HIV viral replication below the detection limit in a selected group of HIV-1-infected individuals.

Age-related M1/M2 phenotype changes in circulating monocytes from healthy/unhealthy individuals.

Macrophage polarization is a candidate biomarker of disease-related inflammatory status, but its modulation during aging has not been investigated. To do this, the M1/M2 profile was assessed by CD80/CD163 gating in classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14lowCD16+) monocytes from 31 healthy subjects (CTRs) of different ages. Cytofluorimetric analysis showed a significantly different CD80/CD163 distribution in the three subsets, as more than 80% of classical and intermediate monocytes were CD80+CD163+, whereas most non-classical monocytes were CD80-CD163- and CD163+. Non-classical CD163+ monocytes were significantly higher whereas classical CD163+ and CD80-CD163- monocytes significantly lower in older than younger CTRs (cut-off, 65 years), suggesting different age-related trends for M2 subsets. To establish whether an M1/M2 imbalance could be associated with disease, 21 patients with acute myocardial infarction (AMI) were compared with older CTRs. The AMI patients showed a significantly decreased proportion of CD163+CD80+ and an increased proportion of CD163+ and CD163-CD80- cells among classical monocytes, opposite trends to those observed in healthy aging. Moreover, a significantly greater proportion of intermediate and non-classical CD80+ monocytes suggested a shift to a pro-inflammatory phenotype. Overall, CD163/CD80 cytofluorimetric characterization of circulating monocytes provides additional information about their polarization and could be an innovative tool to monitor aging.

Impaired in-vitro growth of megakaryocytic colonies derived from CD34 cells of HIV-1-infected patients with active viral replication.

OBJECTIVE: To address the mechanisms of the thrombocytopoietic dysfunction that may follow HIV infection and to compare peripheral blood and bone marrow as sources of CD34 progenitor cells in HIV-infected patients. METHODS: The study used CD34 progenitor cells from 20 previously untreated HIV-infected individuals, 20 HIV-infected individuals treated with antiretroviral therapy and a control group of 20 HIV-uninfected healthy individuals to examine in-vitro megakaryocytopoiesis. There were no hematological abnormalities at baseline in the study groups. CD34 progenitor cells derived from peripheral blood and bone marrow were purified and cultured in medium containing thrombopoietin, interleukin-3, and interleukin-6. HIV-1 plasma viral load was determined by b-DNA technique. Expression of receptors for thrombopoietin, interleukin-3, and interleukin-6 was assessed on CD34 cells by flow cytometry, and numbers of receptors per single cell were calculated by Quanticalc software. RESULTS: Growth of megakaryocytopoietic colony-forming units (CFU-MK) were impaired in untreated HIV-infected individuals despite normal platelet counts. Viral load levels inversely correlate with CFU-MK growth and platelet counts. Antiretroviral drug-treated individuals showed normal megakaryocyte development. Similar results were obtained whether the CD34 progenitor cells derived from peripheral blood or bone marrow. CONCLUSIONS: These findings suggest that megakaryocyte differentiation is impaired before the onset of overt thrombocytopenia in HIV-infected patients and provide evidence for a direct link between viral replication and perturbed megakaryocytopoiesis, which appears to be prevented and/or restored by antiretroviral therapy. The results indicate that peripheral blood represents a suitable source of CD34 hematopoietic progenitors for studies of megakaryocytopoiesis in HIV disease.

Cost of HAART in Italy: multicentric evaluation and determinants from a large HIV outpatient cohort.

BACKGROUND: As HIV infection turned into a chronic treatable disease, now ranking as one of the most costly in medicine, long-term sustainability of highly active antiretroviral treatment (HAART) expenses became a major issue, especially in countries with universal access to care. Identification of determinants of higher HAART costs may therefore help in controlling costs of care, while keeping high levels of retention in care and viral suppression. METHODS: With this aim, we enrolled a large multicentric sample of consecutive unselected human immunodeficiency virus (HIV) patients followed at five sites of care in Italy, and evaluated annual individual HAART costs in relation to a number of sociodemographic, clinical, and laboratory variables. RESULTS: We enrolled 2,044 patients, including 1,902 on HAART. Mean HAART costs were €9,377±€3,501 (range 782-29,852) per year, with remarkable site-based differences, possibly related to the different composition of local assisted populations. Percentages of patients on viral suppression were homogeneously high across all study sites. The factors identified by cross-validation were line of HAART, diagnosis of acquired immune deficiency syndrome, current CD4 T-cell count, and detectable HIV viremia >50 copies/mL. In the final multivariable model, HAART costs were independently directly associated with more advanced HAART line (P<0.001) and inversely correlated with current CD4 T-cell count (P=0.024). Site of care held independent prediction of higher costs, with marked control of expenses at sites 2 (P=0.001) and 5 (P<0.001). CONCLUSION: Higher costs of HAART were strongly associated with previous treatment failures, detectable HIV viremia, and lower CD4 T-cell count at the time of evaluation, with no correlation at all with sex, age, hepatitis C virus coinfection, and nadir CD4 T-cell counts. Newer drugs, which are typically those associated with high prices, at the time of the analysis were still prevalently prescribed to rescue and maintain viral suppression in patients with more complex treatment history. Further analyses of the contribution of the single drug/regimen to the estimated cost are warranted.

Maraviroc as intensification strategy in HIV-1 positive patients with deficient immunological response: an Italian randomized clinical trial.

BACKGROUND: Immunological non-responders (INRs) lacked CD4 increase despite HIV-viremia suppression on HAART and had an increased risk of disease progression. We assessed immune reconstitution profile upon intensification with maraviroc in INRs. METHODS: We designed a multi-centric, randomized, parallel, open label, phase 4 superiority trial. We enrolled 97 patients on HAART with CD4+<200/µL and/or CD4+ recovery ≤ 25% and HIV-RNA<50 cp/mL. Patients were randomized 1:1 to HAART+maraviroc or continued HAART. CD4+ and CD8+ CD45+RA/RO, Ki67 expression and plasma IL-7 were quantified at W0, W12 and W48. RESULTS: By W48 both groups displayed a CD4 increase without a significant inter-group difference. A statistically significant change in CD8 favored patients in arm HAART+maraviroc versus HAART at W12 (p=.009) and W48 (p=.025). The CD4>200/µL and CD4>200/µL + CD4 gain ≥ 25% end-points were not satisfied at W12 (p=.24 and p=.619) nor at W48 (p=.076 and p=.236). Patients continuing HAART displayed no major changes in parameters of T-cell homeostasis and activation. Maraviroc-receiving patients experienced a significant rise in circulating IL-7 by W48 (p=.01), and a trend in temporary reduction in activated HLA-DR+CD38+CD4+ by W12 (p=.06) that was not maintained at W48. CONCLUSIONS: Maraviroc intensification in INRs did not have a significant advantage in reconstituting CD4 T-cell pool, but did substantially expand CD8. It resulted in a low rate of treatment discontinuations. TRIAL REGISTRATION: ClinicalTrials.gov NCT00884858 http://clinicaltrials.gov/show/NCT00884858.

HIV-induced abnormalities in myelopoiesis and their recovery following antiretroviral therapy.

HIV-1 infection is associated with hematologic abnormalities including defective myelopoiesis. Most studies of myelopoiesis during HIV-1 infection were performed using unfractionated bone marrow-derived mononuclear cells, thus resulting in significant inter-individual variability in the numbers of cultured precursors. Here we evaluated the myelopoietic potential of circulating CD34+ progenitors by conducting a longitudinal analysis of antiretroviral therapy (ART)-induced changes of colony forming units-granulocyte and monocyte (CFU-GM) growth. Twelve HIV-infected individuals were studied longitudinally before and after initiation of ART (i.e. at a time when plasma HIV-RNA levels had become undetectable); thirty-one HIV-uninfected healthy individuals were enrolled as controls. Peripheral blood-derived CD34+ progenitors were purified by immunomagnetic sorting, and cultured in methylcellulose-based medium containing stem cell factor, granulocyte-monocyte colony-stimulating factor and interleukin-3. ART-induced changes in the proportion of CD8+ T cells expressing surface HLA-DR were also evaluated. We found that CFU-GM levels were increased in untreated HIV-infected individuals when compared to uninfected controls but declined significantly following ART, in parallel with the decline of HIV-RNA levels in plasma and with the down-regulation of HLA-DR expression on CD8+ T cells. These findings suggest that, in untreated HIV-infected individuals, chronic inflammation and/or immune activation is associated with defective myelopoiesis and accumulation of myeloid precursors. ART-induced suppression of HIV-1 replication is associated with normalization of CFU-GM levels.

Abnormalities of erythropoiesis during HIV-1 disease: a longitudinal analysis.

BACKGROUND: Impaired erythropoiesis is a key abnormality described in untreated HIV-1 disease. Most of the available data on HIV-associated hematopoietic abnormalities were obtained using unfractionated bone marrow-derived mononuclear cells, thus resulting in significant inter (and intra)-individual variability in the number of cultured precursors. Aim of this study was to assess the erythropoietic capability of purified CD34+ progenitors through a longitudinal analysis of burst-forming units-erythroid (BFU-E) growth before and after antiretroviral therapy (ART). METHODS: Twelve HIV-infected individuals were studied before and after ART; 31 HIV-uninfected individuals were enrolled as controls. CD34+ progenitors were purified from peripheral blood by immunomagnetic sorting and cultured in methylcellulose-based medium containing stem cell factor, granulocyte-monocyte colony-stimulating factor, interleukin-3, and erythropoietin. Serum levels of iron, transferrin, transferrin saturation index, soluble transferrin receptor, ferritin, and erythropoietin were also evaluated. RESULTS: Baseline BFU-E levels were increased in untreated HIV-infected individuals when compared with controls but declined significantly after successful ART. In contrast, serum levels of erythropoietin and soluble transferrin receptor increased significantly after ART. CONCLUSIONS: These findings suggest that, in untreated HIV-infected individuals, chronic inflammation and/or immune activation is associated with defective erythropoiesis and accumulation of erythroid precursors. ART-induced suppression of HIV-1 replication is associated with normalization of BFU-E levels.

Cell surface-associated Tat modulates HIV-1 infection and spreading through a specific interaction with gp120 viral envelope protein.

Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transactivator of viral gene expression, has the unusual property of being released by infected cells. Recent studies suggest that extracellular Tat is partially sequestered by heparan sulfate proteoglycans. As a consequence, Tat is concentrated on the cell surface and protected from proteolytic degradation, thus remaining in a biologically active form. We show that Tat binds the surfaces of both HIV-1-infected and surrounding uninfected cells. We provide evidence for a specific interaction between Tat and the HIV-1 glycoprotein 120 (gp120) envelope protein, which enhances virus attachment and entry into cells. We map the interacting sites of both Tat and gp120 and show that synthetic peptides mimicking the gp120 site inhibit HIV-1 infection. Our data demonstrate that membrane-associated Tat is a novel modulator of virus entry and suggest that the Tat-gp120 interaction represents a critical step in HIV-1 spreading during the course of infection.