Validation of a Simple, Rapid, and Cost-Effective Method for Acute Rejection Monitoring in Lung Transplant Recipients.

Despite advances in immunosuppression therapy, acute rejection remains the leading cause of graft dysfunction in lung transplant recipients. Donor-derived cell-free DNA is increasingly being considered as a valuable biomarker of acute rejection in several solid organ transplants. We present a technically improved molecular method based on digital PCR that targets the mismatch between the recipient and donor at the HLA-DRB1 locus. Blood samples collected sequentially post-transplantation from a cohort of lung recipients were used to obtain proof-of-principle for the validity of the assay, correlating results with transbronchial biopsies and lung capacity tests. The results revealed an increase in dd-cfDNA during the first 2 weeks after transplantation related to ischemia-reperfusion injury (6.36 ± 5.36%, p < 0.0001). In the absence of complications, donor DNA levels stabilized, while increasing again during acute rejection episodes (7.81 ± 12.7%, p < 0.0001). Respiratory tract infections were also involved in the release of dd-cfDNA (9.14 ± 15.59%, p = 0.0004), with a positive correlation with C-reactive protein levels. Overall, the dd-cfDNA percentages were inversely correlated with the lung function values measured by spirometry. These results confirm the value of dd-cfDNA determination during post-transplant follow-up to monitor acute rejection in lung recipients, achieved using a rapid and inexpensive approach based on the HLA mismatch between donor and recipient.

Design and numerical simulation of an optofluidic pressure sensor.

We present a numerical design procedure for an all-optical compact sensor by means of integrating the optofluidic switch polymer interferometers to measure the microfluidic air pressure and flow rate. The design is based on a flexible air gap optical cavity that can generate an interference pattern when illuminated by a monochromatic light. The optical interference pattern directly depends on the pressure. In our numerical simulations, we take the effects of fluid flow rate, solid deformation, and the light interference into account. We use the beam propagation method for simulating the optics and the finite element method for simulating the mechanics. The significance of the proposed sensor lies with its low power consumption, compactness, low cost, and short length. This sensor can operate under pressure range of 0-60±6% Pa at a constant temperature of 20 °C.

HLA-DRB1 mismatch-based identification of donor-derived cell free DNA (dd-cfDNA) as a marker of rejection in heart transplant recipients: A single-institution pilot study.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. METHODS: In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. RESULTS: Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. CONCLUSIONS: These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.

Rituximab as a therapeutic tool in severe mixed cryoglobulinemia.

Type II mixed cryoglobulinemia (MC) is a systemic vasculitis, associated in most cases with hepatitis C virus (HCV) infection, sustained by proliferation of oligoclonal cells. Systemic B cell depletion and clinical remission can be achieved in non-Hodgkin lymphoma by human/mouse chimeric monoclonal antibody that specifically reacts with the CD20 antigen (rituximab). Similar effects could be expected in type II MC. Twelve patients, mean age 61.9 years (range 37-76), 11 with HCV infection genotype 2a2c (4 cases) or 1b (6 cases) and 3 (1 case) and symptomatic type II MC with systemic manifestations, including renal involvement, marrow clonal restriction, large necrotizing ulcers, and polyneuropathy, were considered eligible for rituximab therapy because of resistance or intolerance to conventional therapy or important bone marrow infiltration. Rituximab was administered intravenously at a dose of 375 mg/m2 on days 1, 8, 15, and 22. Two more doses were administered 1 and 2 months later. No other immunosuppressive drugs were added. Response was evaluated by assessing the changes in clinical signs, symptoms, and laboratory parameters. Levels of proteinuria, hematuria, erythrocyte sedimentation rate, cryocrit, rheumatoid factor, and IgM decreased while C4 values increased and HCV viral load remained stable during short- and medium-term observation. Bone marrow abnormalities were found to reverse to normal. Constitutional symptoms disappeared or ameliorated. No acute or delayed side effects were seen. Based on this experience and a number of reports published in the last 5 years, Rituximab appears to be a safe and effective therapeutic option in symptomatic patients with HCV-associated MC with signs of systemic vasculitis.

Absence of NF-kappaB activation by a new polystyrene-type adsorbent designed for hemoperfusion.

AIM: The aim of the study was to evaluate biocompatibility of anew polystyrene-type adsorbent (BetaSorb) designed for hemoperfusion, using second-level biomolecular analyses. The device has recently been developed to enhance beta2-microglobulin removal during hemodialysis. Molecular structure and chemical modifications of the surface beads of this cartridge should prevent exposure of dense hydrophobic surface sites to proteins, and avoid the major drawbacks of previous polystyrene-type adsorbent materials. METHODS: Whole blood of healthy donors was incubated in sterile minicolumns packed with BetaSorb Cuprophan, Hemophan, polysulfone and cellulose acetate. In parallel experiments, whole blood was recirculated for 180 min in a sham dialysis circuit equipped with the study sorbent or Hemophan or polysulfone. Biocompatibility was assessed by means of new biomolecular approaches focused on nuclear factor kappaB (NF-kappaB) activation (assessed by electrophoretic mobility shift assay), TNF-alpha and IL-1beta gene expression (evaluated by real-time PCR), TNF-alpha and IL-1beta production (measured by Western blot assay and ELISA), nitric oxide (NO) generation (detected by electron paramagnetic resonance), free oxygen radical production (by chemiluminescence in a biological assay) and the generation of the complement breakdown product C3d. RESULTS: In coincubation experiments, 5-min contact with any dialysis device, but BetaSorb, was enough to induce activation of NF-kappaB. The amount of TNF-alpha precursor form was found to increase after 5 min of exposure to each tested polymer, but no traces of mature forms of TNF-alpha or IL-1beta were detected in in vitro experimental conditions using healthy blood. NO and free oxygen radical generation were significantly lower in blood samples exposed to BetaSorb than in control dialysis devices. C3d levels were found to be increased with Hemophan, unaffected by polysulfone, and remarkably decreased with the BetaSorb device. In the sham hemodialysis experiments, NF-kappaB activation and C3d and NO profiles were similar to direct incubation experiments. Compared to basal levels, quantitation of TNF-alpha and IL-1beta mRNA revealed a 15- and 9-fold increase, respectively, in samples exposed to Hemophan for 180 min. CONCLUSIONS: The new BetaSorb device not only appears to be highly biocompatible, but shares properties that make it probably able to interfere with the activation of the inflammatory state.

Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis.

BACKGROUND: Type II mixed cryoglobulinaemia (MC) is a systemic vasculitis, associated in most cases with hepatitis C virus (HCV) infection, and sustained by proliferation of oligoclonal cells. Systemic B-cell depletion and clinical remission can be achieved in non-Hodgkin lymphoma by a human/mouse chimeric monoclonal antibody that specifically reacts with the CD20 antigen (Rituximab). Similar effects could be expected in type II MC. METHODS: Six patients, mean age 64.2 years (range: 37-76 years), with HCV infection genotype 2a2c (three cases) or 1b (three cases) and symptomatic type-II MC with systemic manifestations, including renal involvement (five cases) and bone marrow clonal restriction (three cases), were considered eligible for Rituximab therapy. Rituximab was administered intravenously at a dose of 375 mg/m(2) on days 1, 8, 15 and 22. Two more doses were administered 1 and 2 months later. No other immunosuppressive drugs were added. Response was evaluated by assessing the changes in clinical signs, symptoms and laboratory parameters for < or = 18 months. RESULTS: Levels of proteinuria, erythrocyte sedimentation rate and cryocrit significantly decreased at 2, 6 and 12 months. Rheumatoid factor and IgM significantly decreased at 6 months whereas C4 values significantly increased at 2 and 6 months. HCV viral load and immunoglobulin G remained stable. Bone marrow abnormalities were found to reverse to normal in all three positive cases. Constitutional symptoms (skin ulcers, purpura, arthralgia, weakness, paraesthesia and fever) disappeared or improved. No acute or delayed side effects were observed. CONCLUSIONS: Rituximab appears to be a safe and effective therapeutic option in symptomatic patients with HCV-associated MC glomerulonephritis and signs of systemic vasculitis.

Nail fold videocapillaroscopy in mixed cryoglobulinaemia.

BACKGROUND: Nail fold videocapillaroscopy (NVC) has been extensively used to examine morphological and functional changes of microcirculation in connective tissue diseases. The nutritional circulation that depends on tissue capillaries, can be expected to be significantly impacted in mixed cryoglobulinaemia (MC). METHODS: Using NVC, we evaluated 29 patients with MC (19 women), mean age 66 years (range 40-83). They included 28 hepatitis C virus (HCV) positive patients-14 genotype 1b, 10 genotype 2a 2c, two genotype 4, two with undetermined genotype. Of them, 18 had type II (IgMk-IgG) MC and 11 had type III. All patients were symptomatic, presenting with weakness (24 of 29 patients), arthralgia (24), purpura (16), peripheral neuropathy (20), Raynaud's phenomenon (8), hypertension (19) and membranoproliferative glomerulonephritis (MPGN) (9). The nail fold capillaries of four fingers per hand were examined using a videomicroscope. RESULTS: Of the 29 patients, 27 had morphological abnormalities (including tortuosity and apical enlargement), 18 had capillaries with deeply altered orientations, 17 had shortened capillaries and 20 neoangiogenetic phenomena. These four types of capillary abnormalities were simultaneously present in 10, suggesting this combination to be a characteristic pattern in MC. Less common alterations included haemorrhages (10 cases), enlarged and giant capillaries (2) and avascular areas (2). The patients with MC-associated MPGN had a significantly greater number of capillary abnormalities (mean 4.5, range 4-6) than non-nephritic patients (mean 3.5, range 1-6, P = 0.01). The number of capillary abnormalities was not related to the presence of Raynaud's phenomenon. Microcirculatory changes did not correlate with other clinical manifestations or serological indices, including cryocrit, cryoglobulin type, HCV genotype, viral load, haemoglobin, ALT, rheumatoid factor, IgM and C4 levels. CONCLUSIONS: Patients with MC show a variety of microcirculatory changes, often clustered in a characteristic pattern of abnormally oriented, short capillaries and neoangiogenetic phenomena. Capillary changes are more numerous in nephritic patients.