Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

Immunoprophylaxis in intensive farming systems: the way forward.

High levels of production in intensive farming systems are associated with increased replacement rates as a result of multifactorial diseases. The so-called "production diseases" may include low-grade infection reducing profitability without increased morbidity. Such infections are sustained by low pathogenic viral and bacterial agents which give rise to full-blown disease in association with poor environmental conditions. In these farms, the results of vaccination may be disappointing. Therefore, fundamental issues should be dealt with toward successful immunoprophylaxis. High lean meat and milk production are associated with chronic inflammation and activation of the innate immune system vis-à-vis cellular stress. This may negatively affect adaptive immune responses. A negative modulation of the host microbiome by farm management practices and drug treatments is a further risk factor. The immune response to stressed cells questions the usual correlates of protection investigated after vaccination. In particular, there is evidence that specific and non-specific immune responses may overlap in vitro as a result of a high level of innate immune responses to Damage-Associated Molecular Patterns (DAMPS) and stress antigens. A vigorous adaptive immune response to microbial agents may be sometimes counterproductive, as suggested in porcine reproductive and respiratory syndrome virus (PRRSV) infection. Alternative outcomes should be sometimes pursued: a better homeostatic control of the inflammatory response, effective and self-limiting innate immune responses, and even tolerance induction. On the whole, successful immunoprophylaxis in intensive farming systems demands co-ordinated and multi-disciplinary efforts in terms of animal breeding, farm management and hygiene, correct choice and harmonization of the prophylactic tools (vaccines, immunomodulators, pre- and probiotics). Finally, there is evidence that disease-predicting parameters of the innate immune response may greatly ease the identification of herds and animals at risk, and contribute to reduced antibiotic usage on farm.

Differential Biological Activities of Swine Interferon-α Subtypes.

Interferons (IFNs) play a crucial role in the host's immune response and other homeostatic control actions. Three IFN types and several IFN families within the types allow for a plethora of regulatory actions. The number of distinct IFN molecules is highest among type I IFNs and, in particular, within the IFN-α family. In pigs, there are 17 IFN-α subtypes with different antiviral activities and different expression profiles; however, no data are available about biological properties other than the antiviral effector activities. Therefore, 16 porcine IFN-α genes were cloned, expressed in mammalian Chinese hamster ovary cells, and characterized for antiviral, anti-inflammatory, and MHC-modulating activities at a pre-established level of 10 IU/mL. Antiviral activity: IFN-α2, -α5, -α9, and -α10 showed the highest level of activity in a pseudorabies virus yield reduction assay. On the contrary, little, if any, activity was shown by IFN-α3, -α7, -α13, -α4, and -α15. Anti-inflammatory activity: With the exception of IFNs-α2, -α7, -α9, and -α11, all IFN-α subtypes had significant anti-inflammatory control activity in an interleukin-8 (IL-8) yield reduction assay. Gene expression analyses showed that some IFN-α subtypes can significantly downregulate the expression of IL-8, tumor necrosis factor α (TNF-α), IL-6, Toll-like receptor 4 (TLR4), ßD1, and nuclear factor-κB (NF-kB) genes, while maintaining or upregulating the expression of ßD4. Immunomodulation: A significant upregulation of class I and/or class II MHC was induced by all the IFNs under study, with the exception of IFNs-α11, -α15, and -α16, which instead significantly downregulated class I MHC. Our results indicate that gene duplications in the porcine IFN-α family underlie diverse effector and regulatory activities, being therefore instrumental in host survival and environmental adaptation. This role of IFN-α could be founded on fine-tuning and regulation of pro- and anti-inflammatory control actions after exposure to both infectious and noninfectious environmental stressors.

Correlates of Protection Following Vaccination with Inactivated Porcine Circovirus 2 Vaccines.

Porcine circovirus type 2 (PCV2) is associated with a number of diseases and syndromes, collectively referred to as porcine circovirus-associated disease. The main objective of this study was to define some in vitro correlates of protection after injection of inactivated PCV2 vaccines with a defined antigen mass. Twelve pigs were vaccinated with three different doses of inactivated, whole-virus antigen (211-844 ng), while four animals were injected with a commercial vaccine (positive control) and four other pigs were mock-vaccinated with phosphate-buffered saline (PBS) in the same oil emulsion. Four weeks later, they were intranasally challenged with 2 × 10(5) TCID50 of a PCV2a strain. Antibody was measured in blood and oral fluids by enzyme-linked immunosorbent assay (ELISA) and a neutralization assay. PCV2 was quantified in serum by real-time polymerase chain reaction for ORF2 gene. PCV2-specific cell-mediated responses were investigated by an IFN-γ release assay in whole blood, IFN-γ ELISPOT, and lymphocyte proliferation (Ki-67 and BrDU assays). All the vaccines under study but mock provided complete or incomplete protection from PCV2 infection in terms of post-challenge viremia. Serum antibody titers (ELISA and neutralizing) after vaccination were not correlated with protection, as opposed to the early neutralizing antibody levels of vaccinated pigs at day 7 after infection. Cell-mediated immune parameters showed a good correlation with vaccine efficacy. In particular, the IFN-γ release assay at 3 weeks after vaccination was an effective marker for predicting protection. All control pigs always tested negative in assays of cell-mediated immunity. Our results outline in vitro testing procedures toward reduced animal usage in the control of PCV2 vaccine batches.

A simple method for measuring porcine circovirus 2 whole virion particles and standardizing vaccine formulation.

Porcine Circovirus 2 (PCV2) is involved in porcine circovirus-associated disease, that causes great economic losses to the livestock industry worldwide. Vaccination against PCV2 proved to be very effective in reducing disease occurrence and it is currently performed on a large scale. Starting from a previous model concerning Foot-and Mouth Disease Virus antigens, we developed a rapid and simple method to quantify PCV2 whole virion particles in inactivated vaccines. This procedure, based on sucrose gradient analysis and fluorometric evaluation of viral genomic content, allows for a better standardization of the antigen payload in vaccine batches. It also provides a valid indication of virion integrity. Most important, such a method can be applied to whole virion vaccines regardless of the production procedures, thus enabling meaningful comparisons on a common basis. In a future batch consistency approach to PCV2 vaccine manufacture, our procedure represents a valuable tool to improve in-process controls and to guarantee conformity of the final product with passmarks for approval. This might have important repercussions in terms of reduced usage of animals for vaccine batch release, in the framework of the current 3Rs policy.

Reduced T-cell repertoire restrictions in abatacept-treated rheumatoid arthritis patients.

BACKGROUND: CD28(neg) T cells, which display functional characteristic of oligoclonally expanded cytotoxic memory T lymphocytes, are believed to be pathologically relevant in rheumatoid arthritis manifestation. The CD28 co-stimulation blockade by abatacept can prevent the generation of CD28(neg) T-cell populations in these patients. METHODS: Samples were obtained before and after 12 months of abatacept therapy. T-cell phenotype and T-cell receptor diversity were evaluated by flow cytometry and complementarity-determining region-3 spectratyping, respectively, while telomerase reverse-transcriptase gene level was measured by real-time PCR. RESULTS: Abatacept induces a decrease of the percentage and number of CD4(+)CD28(neg) T cells and a reduction of T-cell repertoire restrictions; these features are directly correlated. Thymic output and telomerase activity are not modified by the therapy. CONCLUSIONS: Abatacept-induced decrease of peripheral T-cell repertoire restrictions can due to a reduced generation of senescent, chronically stimulated CD4(+)CD28(neg) T cells.

Modulation of Regulatory T-Cell Subsets in Very Long-Term Treated Aviremic HIV(+) Patients and Untreated Viremic Patients.

Naïve, central- and effector-like memory regulatory T cells (Tregs) were evaluated in untreated and long-term antiretroviral-treated HIV(+) patients that showed comparable CD4(+) cell levels, while being, respectively, viremic and aviremic. In the untreated patients, the percentage of naïve-like Tregs was significantly increased to the detriment of central memory regulatory T cells. This redistribution of regulatory Treg subsets may contribute to explain the partially preserved CD4(+) cell counts seen in these patients despite the ongoing viremia. On the contrary, in the long-term treated patients, the percentages of Treg subsets were similar to those of healthy donors, demonstrating a restored Treg homeostasis. The characterization of Treg subsets, rather than an evaluation of the total Treg population, may lead to a deeper understanding of the Treg role in HIV infection and therapy.

Use of V(D)J recombination excision circles to identify T- and B-cell defects and to monitor the treatment in primary and acquired immunodeficiencies.

T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) are circular DNA segments generated in T and B cells during their maturation in the thymus and bone marrow. These circularized DNA elements persist in the cells, are unable to replicate, and are diluted as a result of cell division, thus are considered markers of new lymphocyte output. The quantification of TRECs and KRECs, which can be reliably performed using singleplex or duplex real-time quantitative PCR, provides novel information in the management of T- and B-cell immunity-related diseases. In primary immunodeficiencies, when combined with flow cytometric analysis of T- and B-cell subpopulations, the measure of TRECs and KRECs has contributed to an improved characterization of the diseases, to the identification of patients' subgroups, and to the monitoring of stem cell transplantation and enzyme replacement therapy. For the same diseases, the TREC and KREC assays, introduced in the newborn screening program, allow early disease identification and may lead to discovery of new genetic defects. TREC and KREC levels can also been used as a surrogate marker of lymphocyte output in acquired immunodeficiencies. The low number of TRECs, which has in fact been extensively documented in untreated HIV-infected subjects, has been shown to increase following antiretroviral therapy. Differently, KREC number, which is in the normal range in these patients, has been shown to decrease following long-lasting therapy. Whether changes of KREC levels have relevance in the biology and in the clinical aspects of primary and acquired immunodeficiencies remains to be firmly established.

T-cell Receptor and K-deleting Recombination Excision Circles in Newborn Screening of T- and B-cell Defects: Review of the Literature and Future Challenges.

Since its introduction as a public health programme in the United States in the early 1960s, newborn blood screening (NBS) has evolved from the detection of phenylalanine levels on filter paper to the application of DNA-based technologies to identify T-cell lymphopenia in infants with severe combined immunodeficiency. This latter use of NBS has required the development of an assay for T-cell lymphopenia based on the quantification of T-cell receptor excision circles (TRECs) that could be performed on dried blood spots routinely collected from newborn infants. The TREC-based NBS was developed six years ago, and there have already been 7 successful pilot studies since then. Similarly, efforts are now being made to establish a screen for B-cell defects, in particular agammaglobulinaemia, taking advantage of the introduction of the method for the quantification of K-deleting recombination excision circles (KRECs). A further achievement of NBS could be the simultaneous recognition of T- and B-cell defects using the combined quantification of TRECs and KRECs from Guthrie card blood spots. This approach may help the early identification of infants with T- and B-cell deficiencies so that they can then be referred to specialised paediatric centres, where a precise diagnosis of severe combined immunodeficiency and agammaglobulinaemia can be performed, and where then they can immediately receive specific therapy. Simultaneous TREC and KREC quantification should also allow classification of patients into subgroups and help identify children with less serious primary immunodeficiencies. This would help avoid the opportunistic infections and frequent hospitalisations that result from a late or lack of diagnosis.

Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients.

The anti-α4 monoclonal antibody natalizumab inhibits lymphocyte extravasation into the central nervous system and increases peripheral T and B lymphocytes in multiple sclerosis patients. To investigate whether the lymphocyte accumulation was due to a higher lymphocyte production, an altered homeostasis, or a differential transmigration of lymphocyte subsets through endothelia, T-cell receptor excision circles and kappa-deleting recombination excision circles were quantified before and after treatment, T-cell receptor repertoire was analyzed by spectratyping, and T- and B-lymphocyte subset migration was studied using transwell coated with vascular and lymphatic endothelial cells. We found that the number of newly produced T and B lymphocytes is increased because of a high release and of a low propensity of naïve subsets to migrate across endothelial cells. In some patients this resulted in an enlargement of T-cell heterogeneity. Because new lymphocyte production ensures the integrity of immune surveillance, its quantification could be used to monitor natalizumab therapy safety.

Transforming growth factor-beta1 induces microvascular abnormalities through a down-modulation of neural cell adhesion molecule in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-ß1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-ß1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-ß1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-ß1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-ß1 with anti-TGF-ß1 antibodies or with Ly-364947 (a specific inhibitor TGF-ß1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-ß1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.

Effects of combined antiretroviral therapy on B- and T-cell release from production sites in long-term treated HIV-1+ patients.

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.

Pre-existing T- and B-cell defects in one progressive multifocal leukoencephalopathy patient.

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.

Opposite effects of interferon-ß on new B and T cell release from production sites in multiple sclerosis patients.

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.

MxA RNA quantification in febrile patients who underwent hematopoietic cell transplantation for primary immunodeficiency.

The presence of myxovirus resistance protein A (MxA) RNA was studied in 55 febrile children with primary immunodeficiency, 27 of whom underwent hematopoietic cell transplantation, and in 28 age-matched controls. The level of MxA RNA was above the cutoff, established as the 95th percentile found in controls, with primary immunodeficiency either undergoing transplantation or not in febrile patients, and with a documented diagnosis of infection by adenovirus, cytomegalovirus, Epstein-Barr virus, respiratory syncytial virus, and rotavirus. The presence of rare viral infections, unrecognized among those that more frequently occur in patients with primary immunodeficiency and in patients undergoing transplantation, may explain the high MxA RNA levels observed in some patients with fever but undetectable genomes or antibodies for the more common viruses. The level of MxA in febrile patients with acute graft versus host disease was below the cutoff, with a median level comparable with that observed in patients with primary immunodeficiency, who did not undergo transplantation and were without fever and infections, but significantly lower compared with controls. The level of MxA was well correlated with viral infections in follow-up samples. These data indicate that the measurement of MxA RNA is simple and useful to detect viral infections and in distinguishing them from acute graft versus host disease after allogeneic cell transplantation.

Thymic and bone marrow output in patients with common variable immunodeficiency.

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 different extent of B and T cell immune reconstitution after hematopoietic stem cell transplantation and enzyme replacement therapies in SCID patients with adenosine deaminase deficiency.

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.

Quantification of newly produced B and T lymphocytes in untreated chronic lymphocytic leukemia patients.

BACKGROUND: The immune defects occurring in chronic lymphocytic leukemia are responsible for the frequent occurrence of infections and autoimmune phenomena, and may be involved in the initiation and maintenance of the malignant clone. Here, we evaluated the quantitative defects of newly produced B and T lymphocytes. METHODS: The output of B and T lymphocytes from the production and maturation sites was analyzed in chronic lymphocytic leukemia patients and healthy controls by quantifying kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs) by a Real-Time PCR assay that simultaneously detects both targets. T-lymphocyte subsets were analyzed by six-color flow cytometric analysis. Data comparison was performed by two-sided Mann-Whitney test. RESULTS: KRECs level was reduced in untreated chronic lymphocytic leukemia patients studied at the very early stage of the disease, whereas the release of TRECs+ cells was preserved. Furthermore, the observed increase of CD4+ lymphocytes could be ascribed to the accumulation of CD4+ cells with effector memory phenotype. CONCLUSIONS: The decreased number of newly produced B lymphocytes in these patients is likely related to a homeostatic mechanism by which the immune system balances the abnormal B-cell expansion. This feature may precede the profound defect of humoral immunity characterizing the later stages of the disease.

Transfer of myxovirus-protein-A mRNA assay for interferon-ß bioactivity measurement in multiple sclerosis patients to routine laboratory practice. A 4-year experience.

BACKGROUND: As new biomarkers are validated and their significance in the natural history of specific diseases is established, these technologies can be rapidly transferred to clinical application. Since it has been shown that a single post-interferon-ß (IFNß) injection measurement of myxovirus-protein-A (MxA) mRNA correlates with IFNß bioactivity in IFNß treated patients with multiple sclerosis (MS), we had previously validated an assay for its quantification. METHODS: We introduced a real-time PCR relative quantification assay into routine clinical practice and measured MxA mRNA expression in 564 samples from 500 unselected IFNß treated MS patients over a 4-year period. RESULTS: We confirmed that the assay is reproducible over time, and found that the percentage of patients lacking MxA mRNA induction is comparable to that described in studies performed worldwide after patient selection by pre-screening for the presence of anti-IFNß antibodies. CONCLUSIONS: In view of its simplicity and reproducibility, this MxA assay is an alternative to anti-IFNß antibody determinations for use in routine clinical practice.

Simultaneous quantification of recent thymic T-cell and bone marrow B-cell emigrants in patients with primary immunodeficiency undergone to stem cell transplantation.

A major problem in the field of stem cell transplantation is the difficulty to monitor the efficacy of immune reconstitution. By modifying the widely used method of measuring T-cell receptor excision circles (TRECs) and the recently proposed kappa-deleting recombination excision circles (KRECs) assay, we set up a duplex Real-Time PCR that allowed the simultaneous quantification of newly produced T and B cells in children with primary immunodeficiency undergone to transplantation. We found that lymphocyte recovery involves the mobilization of both new T and B cells from production and maturation sites, and that the increase of TRECs and KRECs can be or strictly associated or independent one from the other. Some patients showed a "lymphocyte rebound" which is followed by a progressive decrease of newly produced T and B lymphocytes starting about 2years after transplantation. In other patients, TRECs and KRECs number remained very low for the entire period of study.