Development and validation of an enzyme linked immunosorbent assay for palivizumab serum determination.

Palivizumab (Synagis) is a humanized monoclonal antibody (IgG1K) composed of 95 percent human and 5 percent murine sequences. It is directed to an epitope in the A antigenic site of the F protein of respiratory syncytial virus (RSV). Palivizumab is used for prevention of serious lower respiratory tract disease caused by RSV in pediatric patients who are at increased risk of severe disease and is administered intramuscularly (IM) for a total of 5 monthly doses. Herein, we report on the development and validation of a very sensitive enzyme-linked immunosorbent assay (ELISA) to measure serum concentrations of palivizumab by a rabbit polyclonal antibody specifically produced against the murine sequence. The method was developed and validated according to the guidelines "Guidance for Industry" (1998) and has proved suitable for the determination of palivizumab serum levels in the target infant population. The ELISA assay was successfully applied to test the serum samples in an infant population who received palivizumab intramuscularly; thus, the assay could be used to determine serum levels in palivizumab-treated infants to optimize dosing and scheduling and to study the relationship between dose and clinical response.

Effects of electromagnetic stimulation on osteogenic differentiation of human mesenchymal stromal cells seeded onto gelatin cryogel.

Bone tissue engineering typically uses biomaterial scaffolds, osteoblasts or cells that can become osteoblasts, and biophysical stimulations to promote cell attachment and differentiation. In this study, we investigated the effects of an electromagnetic wave on mesenchymal stromal cells isolated from the bone marrow and seeded upon gelatin cryogel disks. In comparison with control conditions without electromagnetic stimulus, the electromagnetic treatment (magnetic field, 2 mT; frequency, 75 Hz) increased the cell proliferation and differentiation and enhanced the biomaterial surface coating with bone extracellular matrix proteins. Using this tissue-engineering approach, the gelatin biomaterial, coated with differentiated cells and their extracellular matrix proteins, may be used in clinical applications as an implant for bone defect repair.

Immunomodulatory properties of porcine, bone marrow-derived multipotent mesenchymal stromal cells and comparison with their human counterpart.

Thanks to their immunonodulatory properties, multipotent mesenchymal stromal cells (MSCs) are a promising strategy for preventing/reducing the risk of graft rejection after hematopoietic cell and solid organ transplantation. We have previously demonstrated that porcine MSCs (pMSCs) can be isolated from bone marrow and display similar morphology and differentiative capacity as compared to human MSC (hMSCs). In this study, we investigated the in vitro immunomodulatory properties (namely the ability to suppress lymphocyte proliferation in response to phytohemagglutinin and the cytokine production in the culture supernatants) of pMSCs from six Large White 6-month old piglets. Similarly to hMSCs, pMSCs reduced the phytohemagglutinin-induced lymphocyte proliferation. High levels of IL-6 were found in culture supernatants, whereas IL-10 and TGF-ß were not detectable. In conclusion, ex vivo expanded pMSCs share selected biological/functional properties with hMSCs. pMSCs may be used in in vivo models to investigate novel approaches of prevention of graft rejection in solid organ transplantation.

B lymphocyte subsets and their functional activity in the early months of life.

In the present study we evaluated B-cell subsets and their functional development in 74 newborns from birth to 6 months of life. Moreover, we evaluated natural antibody production in vitro. The results documented a predominance of naive B-lymphocytes at all time-points evaluated, decreasing from birth to 6 months (p=0.009). The percentages of CD27+IgD+ and CD27+IgDneg memory B-cells were very low at birth and significantly increased only at 6 months (p=0.02 and p less than 0.001, respectively). We found a significant increase only in in vitro stimulated IgG production at 6 months as compared to birth (p less than 0.001). Moreover, a lower secretion of anti-Pn IgM antibodies up to 6 months of age, as compared to controls was observed. Our results underline that the susceptibility and severe course of infection in the neonate can be attributed, at least in part, to the lack of pre-existing immunological memory and competent adaptive immunity.

Adipose tissue-derived mesenchymal stromal cells for clinical application: An efficient isolation approach.

PURPOSE OF THE STUDY: Mesenchymal stromal cells (MSCs) are considered a promising tool for cell therapy approaches. The translation of research-based cell culture protocols into procedures that comply with Good Manufacturing Practice (GMP) is critical. The aim of this study was to design a new method for the expansion of MSCs from Adipose Tissue (AT-MSCs) in compliance with GMP, without enzymatic tissue digestion and without the use of animal proteins as source of growth factors. PATIENTS AND METHODS: MSCs were expanded from 10 periumbilical biopsies. Our new isolation approach is based on: (1) disruption of AT with an automated, closed system; (2) use of GMP-grade medium without the addition of fetal bovine serum or platelet lysate; (3) use of human recombinant Trypsin. AT-MSCs cultured in α-MEM and minced by scalpel were used as control. RESULTS: It was possible to expand MSCs from all the AT-samples for at least eight passages. MSCs displayed the typical spindle-shape morphology, a high viability, multilineage differentiation potential and high expression levels of the typical MSC-specific surface antigens and genes. Compared to standard method, MSCs obtained with the new method showed higher yield, up to passage 6, and higher purity in terms of percentage of CD34 and CD45 markers. All AT-MSCs exhibit in vitro immunosuppressive capacity and possess a normal karyotype. CONCLUSIONS: Our data clearly demonstrate that our new approach permits to generate AT-MSCs fully compliant for therapeutic use and better at least in terms of quantity and purity than those obtained with the standard method.

Human colostrum T lymphocytes and their effector cytokines actively aid the development of the newborn immune system.

Colostrum contains soluble and cellular components, the latter mainly T lymphocytes. We expanded in vitro colostrum T lymphocytes (CoTL) to evaluate phenotype and capability of cytokine production. We also considered paired cord blood T-lymphocytes (CBTL) representing the newborn "virgin" immune system. CoTL showed memory phenotype while CBTL expressed mainly naïve phenotype. CoTL included a balanced percentage of helper and cytotoxic subsets. We observed higher percentages of IL-2 (p=0.003) and IL-4 (p=0.027) producing cells by helper rather than by cytotoxic T lymphocytes. The greatest percentage of IFN-gamma producing cells was in cytotoxic cells (p=0.0048), while no difference was found for IL-10. Cord blood samples consisted of a statistically significant greater percentage of helper than cytotoxic cells (p<0.001), with a low percentage of cytokine producing cells, confirming the immaturity of the newborns immune system. CBTL percentage of IL-2 producing cells was higher for helper than cytotoxic subset (p<0.001). We observed a greater percentage of IFN-gamma (p=0.001), IL-4 (p=0.003) and IL-10 (p<0.001) producing cells by cytotoxic than helper T lymphocytes. CoTL demonstrated to protect the newborn through the mothers previous immune experience and to supply active cytokines, which can help the postnatal development of both T type 1/T type 2 response.

A prospective study on children with initial diagnosis of transient hypogammaglobulinemia of infancy: results from the Italian Primary Immunodeficiency Network.

Transient hypogammaglobulinemia of infancy (THI) is a heterogeneous disorder characterized by reduced serum IgG levels in early infancy. A putative diagnosis is initially made after exclusion of other causes of hypogammaglobulinemia while a definitive diagnosis of THI can only be made a posteriori in patients with normalization of IgG levels. The aim of this study is to characterize clinical and immunological features of children with an initial diagnosis of THI in correlation to natural outcome, and to assess predictive laboratory parameters of clinical evolution for this disorder. We prospectively analysed clinical and immunological characteristics of 77 THI children at initial diagnosis and of 57 patients at follow-up. Memory B cell subsets and in vitro immunoglobulin production were evaluated. Seventy patients (91 percent) showed clinical symptoms. Patients suffered from infections (91 percent), allergies (47 percent) and autoimmune disease (4 percent). During follow-up 41/57 children (72 percent) normalized IgG values, mostly within 24 months of age (p less than 0.001), allowing the diagnosis of THI. The 16 children who did not normalize their IgG levels showed a higher frequency of severe infections and autoimmune disease (p less than 0.01). Moreover, they expressed a reduced frequency of IgM and switched memory B cells (p less than 0.01) and an inability to produce IgG in vitro (p less than 0.02). We conclude that most patients with an initial diagnosis of THI spontaneously recover within 24 months of age and have a benign clinical course, while a subgroup of children with undefined hypogammaglobulinemia share a clinical and immunological profile with other primary immunodeficiencies. Early recognition of children with hypogammaglobulinemia during infancy who are likely to suffer from permanent immunodeficiencies later in life would allow prompt and appropriate laboratory and clinical interventions.

Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

Escherichia coli specific secretory IgA and cytokines in human milk from mothers of different ethnic groups resident in northern Italy.

Breast milk supplies many bioactive components. Neonates protection from pathogenic bacteria is mainly attributable to secretory IgA antibodies present in human milk in an amount depending on previous antigenic exposure. To bring new details into the field of immunological memory in secretory immunity, we evaluated the production of s-IgA specific for E. coli (E. coli s-IgA), and of pro-inflammatory (IL-6 and IL-8) or anti-inflammatory (IL-10) cytokines in the milk of mothers of different ethnic groups exposed in the past to poor conditions, but nowadays living in Italy in adequate conditions. Mothers from Italy, Africa, Asia and Eastern European Countries were included in the study. Anti-E. coli s-IgA, IL-6, IL-8 and IL-10 were determined by ELISA. Breast milk of all the foreign mothers presented higher levels of E. coli s-IgA than Italians, and for Asian and African mothers were significative (p=0.031 and p=0.015, respectively). Milk from women of Eastern European Countries revealed the highest IL-8 levels (p=0.026), while milk from Asian women presented the greatest concentration of IL-6 (p=0.04); however, the Africans reported the lowest concentrations of IL-10 (p=0.045). Since all the mothers had been living in Italy for some time, we believe that the presence of high levels of E.coli s-IgA, supported by high levels of pro-inflammatory cytokine, is part of a persisting immunological secretory memory.

A new sensitive enzyme-linked immunosorbent assay (ELISA) for Alemtuzumab determination: development, validation and application.

Alemtuzumab is a humanized (IgG(1)) rat monoclonal antibody to CD52 antigen and is currently used in the treatment of chronic lymphocytic leukemia (CLL) and other CD52-positive lymphoproliferative disorders. Various techniques have been developed to measure Alemtuzumab levels in human serum/plasma. The authors report on the validation of a very sensitive enzyme-linked immunosorbent assay (ELISA) to measure serum concentrations of the humanized IgG(1) using a rabbit polyclonal antibody specifically produced against the rat sequence of Alemtuzumab after papain digestion. The assay was successfully applied to test the serum samples of patients with B-lymphocyte CLL who received Alemtuzumab subcutaneously. This ELISA assay could be easily used to determine human serum levels of Alemtuzumab pre- and post-treatment to optimize dosing and scheduling and to study the relationship between dose and clinical response.

Deficiency of INFgamma producing cells in adenoids of children exposed to passive smoke.

Exposure to passive smoke is a very common event associated with increased susceptibility to respiratory tract infections. Many related adverse effects result from the ability of cigarette smoke extracts to interfere with the immune system, but the mechanism is not yet completely understood. The aim of the present study is to evaluate the intracellular cytokine profile in adenoids and peripheral blood cells of children exposed to passive smoke. Children undergoing adenoidectomy exposed or not exposed to passive smoke were studied. The intracellular cytokine profile of lymphocyte subsets in adenoids and in peripheral blood were evaluated by flow cytometry analysis. Children exposed to tobacco smoke showed a significantly lower percentage of INF-gamma producing CD4+ and CD8+ cells in adenoids. Moreover a significant correlation was observed between the quantity of exposure and reduction in Th1 (CD4+INFgamma+ and CD8+INFgamma+) cells in adenoids. This reduction may be a contributing factor in the increasing susceptibility to respiratory tract infection in children exposed to tobacco smoke.

The immunosuppressive effect of human cytomegalovirus infection in recipients of allogeneic hematopoietic stem cell transplantation.

In immune-competent individuals, human cytomegalovirus (HCMV) infection is associated with impairment of T-cell function. Our goal was to evaluate prospectively whether clinically asymptomatic HCMV infection in allogeneic hematopoietic stem cell transplantation (alloHSCT) recipients, treated pre emptively with ganciclovir, influences T-cell function as well. Mitogen-stimulated T-cell proliferative activity, together with cell surface markers, was tested in 49 patients on days + 30, + 45, + 60, and + 90 after alloHSCT and, additionally, in cases of positive HCMV pp65-antigenemia. HCMV infection was diagnosed in 19 patients. None of them developed HCMV disease. T-cell proliferative activity was significantly decreased on days when HCMV antigenemia was positive as compared to days without antigenemia. The number of pp65-positive cells negatively correlated with proliferative response. Comparison of patients who did experience HCMV infection with those who did not reveals significant decrease of T-cell proliferative activity observed on days + 30 and + 45, a time period when antigenemia was most frequently found to be positive, whereas no difference was detected on days + 60 and + 90. We conclude that, even clinically asymptomatic, HCMV infection has negative impact on T-cell proliferation capacity in alloHSCT recipients. However, pre emptive therapy with ganciclovir makes this immunosuppressive effect transient and restricted to the time of infection duration.

IFN-gamma low production capacity in type 1 diabetes mellitus patients at onset of disease.

In type 1 diabetes mellitus (T1DM), cytokines can be directly cytotoxic to beta-cells, and/or play an indirect role influencing some cells of the immune system. Since several factors could impair cytokine serum levels, the purpose of our study was to longitudinally evaluate intracellular cytokines, in T1DM patients, and in subject at risk, by flow cytometry analysis. At T1DM onset we observed significantly lower percentage of peripheral CD4 + and CD8 + cells producing IFN-gamma in patients compared to controls and subjects at risk. The 15-month follow-up patients showed significantly lower percentage of CD4 + and CD8 + cells producing IFN-gamma compared to the other groups. At 8-year follow-up no significant differences were observed among the groups in the percentage of cells producing cytokines. We could have considered "exhausted cells" or these T cell subsets may be migrated from peripheral blood to pancreas. On the other hand, our results are in agreement with those reported in literature: in animal model the absence of IFN-gamma production makes beta-cells highly susceptible to viral infection and subsequent attack by natural killer cells, which lead to hyperglycaemia and diabetes mellitus.

Immunoglobulin G3-specific antibodies as a marker for early diagnosis of HIV infection in children.

Early diagnosis of HIV infection in the child of an HIV-infected mother may be difficult as HIV-specific immunoglobulin (Ig) G antibodies are transmitted to the fetus transplacentally. In an attempt to provide a new, simpler tool for early identification of HIV-infected children we analysed the HIV-specific IgG subclass pattern during the first year of life. One hundred and one samples were collected from 35 children born to HIV-seropositive mothers, among whom 18 seroreverted during follow-up and 17 were HIV-infected (two P1 and 15 P2 according to the Centers for Disease Control classification). Serum HIV-specific IgG3 was detectable at least in one sample in 26 out of 35 children. All 17 HIV-infected children showed persistently detectable specific IgG3, both with stable or progressive disease. Out of the 18 uninfected children who seroreverted during follow-up, nine were HIV-specific IgG3-negative when first tested and nine lost HIV-specific-IgG3 within 28 weeks after birth. The correlation of the serological results with clinical information and any other diagnostic tool on each child suggests that the clearance of specific-IgG3 antibodies heralds seroconversion in uninfected passive antibody-carrier children. This observation provides the basis for a new, simple and effective method for early diagnosis of HIV infection in children born to seropositive mothers.

An avidin-biotin ELISA for the measurement of serum and secretory IgD.

This paper describes an improved microtiter solid-phase enzyme immunoassay for the determination of serum and secretory IgD. Use of the interaction between biotinylated anti-human IgD and horseradish peroxidase(HRP)-avidin conjugate permits quantitation of human IgD in the range of 1-64 ng/ml. IgD was detected in all samples of serum, saliva and nasal secretions of 28 normal adults. In only one subject both serum and secretory IgD were undetectable. The mean concentration of serum IgD determined by this assay is similar to that reported by other authors using radioimmunoassay. The assay described is not only rapid and inexpensive but at least as sensitive as the radioimmunoassays usually employed for quantitation of IgD.

Elevated IgA anti-gliadin antibodies in juvenile chronic arthritis.

Increased intestinal permeability secondary to treatment with non-steroidal anti-inflammatory drugs (NSAIDs) and raised levels of anti-gliadin antibodies (AGA) have been reported in adults with rheumatoid arthritis. We have therefore retrospectively investigated the presence of serum AGA of the IgA and IgG classes in 70 patients with juvenile chronic arthritis (JCA). Serum IgA (but not IgG) AGA were found to be higher in JCA patients than in controls (6.2 +/- 8.7 vs 2.1 +/- 1.5 AU/ml; p less than 0.0001). This finding was observed independently of the JCA onset subtype or disease activity; however, lower levels of IgA AGA were found in patients with pauciarticular JCA and in those in remission. No significant differences in IgA AGA serum levels were observed between untreated patients and patients treated with NSAIDs. Five patients who presented the highest levels of IgA AGA were further studied a second time; serum IgA AGA were found to be markedly reduced or normalized and no clinical or laboratory evidence of coexistent coeliac disease was observed. In conclusion, our results suggest that the elevation of IgA AGA seen in our patients is secondary to non-specific immune stimulation rather than to an NSAID-induced increase in intestinal permeability.

Celiac disease and type I (insulin-dependent) diabetes mellitus in childhood: follow-up study.

To ascertain the specificity of IgA and IgG antigliadin (IgA-AGA, IgG-AGA), IgA-antireticulin (R1-ARA), and antiendomysial (AEA) antibodies for the diagnosis of celiac disease, we evaluated 133 type I diabetic children aged 1.4-28.4 years (mean 14.1 +/- 6.6), with diabetes from onset to 20.5 years. Fifty-three patients were considered at onset and 49 of these also during follow-up. IgA-AGA and IgG-AGA were determined by enzyme-linked immunosorbent assay (ELISA), R1-ARA and AEA by indirect immunofluorescence. IgA-AGA were positive in 20 of 133 (15%), IgG-AGA were positive in seven of 133 (5.26%), while R1-ARA and AEA were positive in three patients. At the onset of disease we found elevated IgA-AGA in 17 of 53 (32%) patients, IgG-AGA in four (7.55%) patients, three of them with IgA-AGA as well; R1-ARA and AEA were present in three (5.66%) patients, all with high IgA-AGA levels. During 1-10 year follow-up IgA-AGA decreased to within the normal range in 13 patients, with elevated IgA-AGA at onset but without R1-ARA and AEA; in four patients with high IgA-AGA at onset, IgA-AGA remained constantly elevated as did R1-ARA and AEA in three of them; and two patients, without IgA-AGA, R1-ARA, and AEA at onset, became positive for all three antibodies. Intestinal biopsy confirmed a diagnosis of celiac disease in five of these with IgA-AGA, R1-ARA, and AEA, but not in one patient with persistent IgA-AGA but no AEA and R1-ARA, suggesting that R1-ARA and AEA are more reliable markers for the screening of celiac disease in type I diabetic patients.

Clinical aspects of coeliac disease in children with insulin-dependent diabetes mellitus.

Coeliac disease (CD) is heterogeneous in its clinical presentation and pathological expression. Silent, latent and potential forms represent the submerged part of the so-called "coeliac iceberg". The association of insulin-dependent diabetes mellitus (IDDM) and CD has been widely reported. For the screening of CD in diabetic patients, anti-reticulin R1 (ARA-R1) and anti-endomysium (AEA) antibodies are more reliable markers than anti-gliadin (AGA) antibodies. Recent studies have reported an increased prevalence of CD in children with IDDM. In our experience intestinal biopsy confirmed a diagnosis of CD in 6 out of 172 diabetic patients, with a prevalence of 3.5%. Only occasionally does CD precede the onset of IDDM; more often CD is diagnosed shortly or sometimes years after the onset of diabetes. Typical gastrointestinal complaints of CD (such as diarrhoea, abdominal distension) are rare in IDDM patients, while atypical isolated signs or symptoms of CD are more common, in particular sideropenic anemia, short stature, delayed puberty, epilepsy, hypertransaminasemia, dyspeptic symptoms, herpetiform dermatitis, and recurrent aphthous stomatitis. It is recommended that all diabetic children, even those asymptomatic, should be screened yearly for CD, using a combination of AGA plus ARA-R1 and AEA.

Co-infusion of ex vivo-expanded, parental MSCs prevents life-threatening acute GVHD, but does not reduce the risk of graft failure in pediatric patients undergoing allogeneic umbilical cord blood transplantation.

When compared with BMT, umbilical cord blood transplantation (UCBT) is associated with a lower rate of engraftment and delayed hematological/immunological recovery. This leads to increased risk of TRM in the early post transplantation period due to infection. Acute GVHD, although occurring less frequently in UCBT compared with BMT, is also significantly associated with increased rate of early TRM. BM MSCs are known to support normal in vivo hematopoiesis, and co-transplantation of MSCs has been shown to enhance engraftment of human cord blood hematopoietic cells in nonobese diabetic/SCID mice. In 13 children with hematological disorders (median age 2 years) undergoing UCBT, we co-transplanted paternal, HLA-disparate MSCs with the aim of improving hematological recovery and reducing rejection. We observed no differences in hematological recovery or rejection rates compared with 39 matched historical controls, most of whom received G-CSF after UCBT. However, the rate of grade III and IV acute GVHD was significantly decreased in the study cohort when compared with controls (P=0.05), thus resulting in reduced early TRM. Although these data do not support the use of MSCs in UCBT to support hematopoietic engraftment, they suggest that MSCs, possibly because of their immunosuppressive effect, may abrogate life-threatening acute GVHD and reduce early TRM.