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Background: The immune system reconstitution in HIV-1- infected patients undergoing combined antiretroviral therapy is routinely evaluated by T-cell phenotyping, even though the infection also impairs the B-cell mediated immunity. To find new laboratory markers of therapy effectiveness, both B- and T- immune recovery were evaluated by means of a follow-up study of long-term treated HIV-1- infected patients, with a special focus on the measure of new B- and T-lymphocyte production., Methods: A longitudinal analysis was performed in samples obtained from HIV-1-infected patients before therapy beginning and after 6, 12, and 72 months with a duplex real-time PCR allowing the detection of K-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs), as measures of bone-marrow and thymic output, respectively. A cross sectional analysis was performed to detect B- and T-cell subsets by flow cytometry in samples obtained at the end of the follow-up, which were compared to those of untreated HIV-1-infected patients and uninfected controls., Results: The kinetics and the timings of B- and T-cell release from the bone marrow and thymus during antiretroviral therapy were substantially different, with a decreased B-cell release and an increased thymic output after the prolonged therapy. The multivariable regression analysis showed that a longer pre-therapy infection duration predicts a minor TREC increase and a major KREC reduction., Conclusions: The quantification of KRECs and TRECs represents an improved method to monitor the effects of therapies capable of influencing the immune cell pool composition in HIV-1-infected patients.

Progressive multifocal leukoencephalopathy (PML) usually occurs in patients with severe immunosuppression, hematological malignancies, chronic inflammatory conditions or receiving organ transplant. Recently, PML has also been observed in patients treated with monoclonal antibodies. By taking advantage of the availability of samples from a multiple sclerosis (MS) patient treated with natalizumab, the antibody anti-α4 integrin, who developed PML and was monitored starting before therapy initiation, we investigated the fate of T and B lymphocytes in the onset of PML. Real-time PCR was used to measure new T- and B-cell production by means of T-cell receptor excision circle (TREC) and K-deleting recombination excision circle (KREC) analysis and to quantify transcripts for CD34, terminal-deoxynucleotidyltransferase, and V pre-B lymphocyte gene 1. T- and B-cell subsets and T-cell heterogeneity were measured by flow cytometry and spectratyping. The data were compared to those of untreated and natalizumab-treated MS patients and healthy donors. Before therapy, a patient who developed PML had a low TREC and KREC number; TRECs remained low, while KRECs and pre-B lymphocyte gene 1 transcripts peaked at 6 months of therapy and then decreased at PML diagnosis. Flow cytometry confirmed the deficient number of newly produced T lymphocytes, counterbalanced by an increase in TEMRA cells. The percentage of naive B cells increased by approximately 70% after 6 months of therapy, but B lymphocyte number remained low for the entire treatment period. T-cell heterogeneity and immunoglobulins were reduced. Although performed in a single patient, all results showed that an immune deficit, together with an increase in newly produced B cells a few months after therapy initiation, may predispose the patient to PML. These findings indicate the TREC/KREC assay is a potential tool to identify patients at risk of developing PML and may provide insights into the immunological involvement of monoclonal antibody-associated therapies.

The release of newly produced B and T lymphocytes from the production sites was analyzed in 30 multiple sclerosis patients treated with interferon-beta by measuring T-cell receptor excision circles and k-deleting recombination excision circles. We found that the therapy induces opposite effects on B- and T-cell mobilization in 33% of patients. New B-cell production, which peaks after 6 months of therapy and then decreases to levels that, however, are still higher than in controls, may cause a renewal of the B-cell compartment. On the contrary, the decreased number of newly produced T lymphocytes observed at 12 months of treatment and the association between reduced thymic output and low peripheral T lymphocytes can be a cause of leukopenia, a frequent side effect of the therapy., (Copyright © 2011. Published by Elsevier B.V.)

Objective: The study aims to obtain more information about the immune deficit of common variable immunodeficiency (CVID) patients., Materials and Methods: A new real-time PCR assay was used to quantify T and B lymphocyte mobilization from the production and maturation sites through the detection of T cell receptor excision circles (TRECs) and kappa-deleting recombination circles (KRECs) and to allow the estimation of the average number of B cell divisions. T and B lymphocyte subsets were analyzed by flow cytometry., Results: The number of TREC(+) lymphocytes, which depends on age and gender, was significantly reduced in CVID patients. Similarly, KREC concentration was lower than in controls. Classification of patients according to the percentage of memory switched B cells showed that patients belonging to MB2 group and therefore with conserved B cell maturation have the lowest new B cell output but increased average peripheral divisions, leading to the highest B cell number., Conclusions: TREC and KREC quantification can be helpful for a more complete and informative understanding of a heterogeneous disease such as CVID.

The lack of adenosine deaminase (ADA) leads to the accumulation of toxic metabolites, resulting in SCID. If the disease is left untreated, it is likely to have a fatal outcome in early infancy. Because hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy with pegylated bovine ADA (PEG-ADA) are both provided in our hospital, we undertook a retrospective longitudinal comparative study of the extent of lymphocyte recovery in two groups of treated ADA-SCID children. Together with classical immunological parameters, we quantified the output of the new B and T cells from the production sites using the κ-deleting recombination excision circle and TCR excision circle assay, and we monitored T cell repertoire diversification. We found that immune reconstitution was different following the two treatments. The stable production of κ-deleting recombination excision circle(+) lymphocytes sustained an increase in B cell number in HSCT-treated patients, whereas in PEG-ADA-treated patients, it was accompanied by a significant and progressive decrease in circulating CD19(+) lymphocytes, which never reached the levels observed in age-matched children. The mobilization of TCR excision circle(+) cells, though lower than in controls, was stable with time after HSCT treatment, leading to a constant peripheral T cell number and to the diversification of the T cell repertoire; however, it was compromised in children receiving prolonged PEG-ADA therapy, whose T cells showed progressively narrowing T cell repertoires.

Combination of chemotherapy and immunotherapy to increase the effectiveness of an antitumor immune response is currently regarded as an attractive antitumor strategy. In a pilot clinical trial, we have recently documented an increase of melanoma antigen A (Melan-A)-specific, tumor-reactive, long-lasting effector-memory CD8(+) T cells after the administration of dacarbazine (DTIC) 1 day before peptide vaccination in melanoma patients. Global transcriptional analysis revealed a DTIC-induced activation of genes involved in the immune response and leukocyte activation. To identify the possible mechanisms underlying this improved immune response, we have compared the endogenous and the treatment-induced anti-Melan-A response at the clonal level in patients treated with the vaccine alone or with DTIC plus vaccine. We report a progressive widening of T-cell receptor (TCR) repertoire diversity, accompanied by high avidity and tumor reactivity, only in Melan-A-specific T-cell clones of patients treated with chemoimmunotherapy, with a trend toward longer survival. Differently, patients treated with vaccine alone showed a tendency to narrowing the TCR repertoire diversity, accompanied by a decrease of tumor lytic activity in one patient. Collectively, our findings indicate that DTIC plus vaccination shapes the TCR repertoire in terms of diversity and antitumor response, suggesting that this combined therapy could be effective in preventing melanoma relapse., (©2010 AACR.)

A genotyping assay was setup to assess the prevalence, in the population of a Northern Italian city, of the C/T-13910 single nucleotide polymorphism, closely associated to lactose malabsorption in many world areas including Sardinia. The results were compared to published Italian data, in order to evaluate the worth of a future validation of the assay for use in routine practice. DNA was extracted from blood samples of 123 randomly chosen healthy blood donors coming from the same city area, and was analyzed by a real-time polymerase chain reaction (PCR) genotyping assay; the frequency of the hypolactasia-associated CC-genotype was compared to the weighted average of results extracted from studies reporting the frequency of hypolactasic phenotype or genotype in nearby or distant Italian regions. Sixty-five percent of donors carried the CC-genotype, a percentage similar to other northern Italian cities, but significantly higher than what previously determined in surrounding Italian regions at the phenotype level, i.e. by breath test. This discrepancy parallels recent reports of non concordance between results of genotyping and hypolactasic phenotype in some world areas, including a neighbouring Northern Italian city. A north-south gradient of CC-prevalence was also observed. These results reinforce the notion of wide inter-regional variations in the frequency of C/T-13910 polymorphism and of incostant concordance with hypolactasic phenotype, even in subjects from the same country. Given the unsatisfactory results recently obtained from validation of a related assay in a neighbouring city, the authors decided not to proceed further and keep the assay only as a diagnostic aid in special situations.

Interferon-beta receptor (IFNAR) is composed of 2 subunits, IFNAR1 and IFNAR2, the latter of which is expressed as functional (IFNAR2.2), non-functional (IFNAR2.1) and soluble (IFNAR2.3) isoform. Real-Time PCR analysis of mRNA for all IFNAR components in multiple sclerosis patients naïve for therapy and undergoing long-term treatment with interferon-beta shows that IFNAR1 mRNA level is lower than in healthy controls. If long-term treated patients are divided according to the production of mRNA for Myxovirus protein-A, a marker of interferon-beta bioactivity, IFNAR1 mRNA reaches the values observed in controls only in Myxovirus protein-A-induced patients. Since chronic cell stimulation by interferon-beta induces IFNAR protein down-regulation, we suggest that the increase of IFNAR1 mRNA might serve as a mechanism for counterbalancing the loss of protein receptor, enhancing, at least in this sub-group of patients, cell responsiveness to interferon-beta.

We developed a real-time PCR assay to simultaneously measure the mRNA level of type I interferon (IFN) receptor (IFNAR) components in peripheral blood cells of children with chronic immune stimulation due to HIV infection. All patients were undergoing antiretroviral therapy and were divided into two groups on the basis of the induction of MxA mRNA, a marker of type I IFN bioactivity. We found that IFNAR-2 subunit mRNA was higher than that of the IFNAR-1 subunit, that the mRNA for the IFNAR-2.2 functional isoform was more expressed than that for the truncated IFNAR-2.1 isoform, and both were much more represented than that of the IFNAR-2.3 soluble isoform. We also demonstrated that soluble isoform mRNA was significantly diminished in the subgroup of patients with MxA mRNA below the cutoff value (determined as the 99th percentile of MxA measured in healthy controls). These results suggest that downregulation of the soluble receptor isoform, which would not compete with the functional isoform for binding to the target cytokine, would give type I IFN, eventually induced in these patients in the case of viral reactivation, the opportunity to promptly exert its antiviral activity.