Roberto Paganelli

Pathogenesis of tendinopathies: inflammation or degeneration?

The intrinsic pathogenetic mechanisms of tendinopathies are largely unknown and whether inflammation or degeneration has the prominent role is still a matter of debate. Assuming that there is a continuum from physiology to pathology, overuse may be considered as the initial disease factor; in this context, microruptures of tendon fibers occur and several molecules are expressed, some of which promote the healing process, while others, including inflammatory cytokines, act as disease mediators. Neural in-growth that accompanies the neovessels explains the occurrence of pain and triggers neurogenic-mediated inflammation. It is conceivable that inflammation and degeneration are not mutually exclusive, but work together in the pathogenesis of tendinopathies.

Unravelling the Complexity of T Cell Abnormalities in Common Variable Immunodeficiency

We investigated several phenotypic and functional parameters of T cell-mediated immunity in a large series of common variable immunodeficiency (CVID) patients. We demonstrated that the vast majority of CVID patients presented multiple T cell abnormalities intimately related among them, the severity of which was reflected in a parallel loss of CD4+ naive T cells. A strong correlation between the number of CD4+ naive T cells and clinical features was observed, supporting the subgrouping of patients according to their number of naive CD4+ T lymphocytes. A reduced thymic output and disrupted CD4+ and CD8+ TCR repertoires paralleled the contraction of CD4+ naive T cell pools. The evaluation of activation markers and cytokine production indicated a strong T cell activation that was significantly related to the increased levels of T cell turnover and apoptosis. Finally, discrete genetic profiles could be demonstrated in groups of patients showing extremely diverse T cell subset composition and function. Naive CD4+ T cell levels were significantly associated with the switched memory B cell-based classification, although the concordance between the respective subgroups did not exceed 58.8%. In conclusion, our data highlight the key role played by the T cell compartment in the pathogenesis of CVID, pointing to the need to consider this aspect for classification of this disease.

Allergenicity of goat’s milk in children with cow’s milk allergy

BACKGROUND Cows milk allergy (CMA) is a common disease of infancy and childhood. An appropriate cows milk (CM) substitute is necessary for feeding babies with CMA. CM substitutes are soy formulas and casein- or whey-based extensively hydrolyzed formulas. In several countries, including Italy, goats milk (GM) formulas are available, and some physicians recommend them for feeding babies with CMA. OBJECTIVE We sought to investigate, in vitro and in vivo, the allergenicity of GM in 26 children with proven IgE-mediated CMA. METHODS All the children underwent skin tests with CM and GM; detection of specific serum IgE to CM and GM; and double-blind, placebo-controlled, oral food challenges (DBPCOFCs) with fresh CM, GM, and, as placebo, a soy formula (Isomil, Abbott, Italy). CAP inhibition and immunoblotting inhibition assays were also carried out in 1 of 26 and 4 of 26 children with positive RAST results to both CM and GM, respectively. RESULTS All the children had positive skin test responses and CAP results to both CM and GM, all had positive DBPCOFC results to CM, and 24 of 26 had positive DBPCOFCs to GM. In CAP inhibition tests, preincubation of serum with CM or GM strongly inhibited IgE either to CM or to GM. In immunoblotting inhibition assays, preincubation with CM completely extinguished reactivity to GM, whereas GM partially inhibited reactivity to CM. CONCLUSIONS These data strongly indicate that GM is not an appropriate CM substitute for children with IgE-mediated CMA. A warning on the lack of safety of GM for children with CMA should be on the label of GM formulas to prevent severe allergic reactions in babies with CMA.

Changes in circulating B cells and immunoglobulin classes and subclasses in a healthy aged population

The study of 87 adults of different ages, including 15 centenarians, selected for their healthy status, showed that profound changes of humoral immunity occur throughout life. In particuIar, a statistically significant age‐reIated increase of the serum level of immunoglobulin cIasses (IgG and IgA but not IgM) and IgG subcIasses (IgGI, 2 and 3, but not IgG4) was detected. A parallel age‐related decrease of circuIating B cells was also observed. The hypothesis of a complex derangement of B cell function and/or compartmentalization with age is put forward, together with the proposal that healthy centenarians (as representative of successful ageing) may be helpful in identifying the physiological age‐reIated modifications of the immune system.

Long-term evaluation of T-cell subsets and T-cell function after HAART in advanced stage HIV-1 disease.

OBJECTIVES Evaluation of immunological reconstitution after 2 years of highly active antiretroviral therapy (HAART) in AIDS patients. DESIGN Previous data showed the effectiveness of HAART but conflicting evidence of immune reconstitution has been found in severely immunocompromised patients. Therefore, T-cell subsets and functions were analysed during 24 months of HAART in 21 AIDS patients (mean baseline CD4 cell count, 20 x 10(6)/l). METHODS Subjects were tested at baseline and after 4, 12 and 24 months of therapy for clinical symptoms and the following investigations were carried out: plasma HIV RNA, T-cell subsets and lymphoproliferative responses to mitogens (phytohaemagglutinin, anti-CD3), and recall antigens (Candida mannoprotein, tetanus toxoid and recombinant glycoprotein 160). RESULTS Increase in body weight, improvement of Karnofskys score and reduction of opportunistic infections were observed. All patients showed an initial increase in the CD4 memory subset, whereas naive CD4 cells consistently increased only after 1 year. The magnitude of immune recovery was stronger in patients showing a significant reduction in viral load. However seven out of 21 patients who did not reach a sustained suppression of viral load showed also an increase in T-cell subsets. The majority of patients recovered lymphoproliferative responses to mitogens, whereas only four subjects showed a functional response to Candida mannoprotein. No patients showed a response to HIV recombinant glycoprotein 160 or tetanus toxoid. CONCLUSIONS The immune recovery observed is slower and not complete in severely immunocompromised patients. Our data suggest that HAART may be continued also in the absence of a significant HIV RNA decrease if alternative drugs are not available.

Extremely low frequency pulsed electromagnetic fields increase interleukin-2 (IL-2) utilization and IL-2 receptor expression in mitogen-stimulated human lymphocytes from old subjects

The effects of the exposure of mitogen‐stimulated human lymphocytes from aged subjects to low‐frequency pulsed electromagnetic fields (PEMFs) were studied by measuring the production of interleukin‐2 (IL‐2) and the expression of IL‐2 receptor. PEMF‐exposed cultures that presented increased [3H]thymidine incorporation showed lower amounts of IL‐2 in their supernatants, but higher percentages of IL‐2 receptor‐positive cells and of T‐activated lymphocytes. Taken together, these data suggest that PEMFs were able to modulate mitogen‐induced lymphocyte proliferation by provoking an increase in utilization of IL‐2, most likely acting on the expression of its receptor on the plasma membrane.

Chemokines, sTNF-Rs and sCD30 serum levels in healthy aged people and centenarians.

Several lines of evidence point to a profound remodelling of the cytokine network in healthy elderly subjects, with decreased type-1 cytokine production (IL 2) and a shift to type 0 and 2. We have also observed an increase of proinflammatory cytokines (IL-1, IL-6, TNF-alpha) in vitro, and an increase of circulating stem cell factor in vivo. In this setting, we studied changes of chemokines (MCP-1 and RANTES) with aging, as well as other molecules, namely, sTNF-RI and sTNF-RII, and the soluble form of the CD30 molecule (sCD30), involved in the pro- and antiinflammatory cytokine balance. The subjects enrolled in the study belonged to three different selected healthy groups of young, aged and centenarians. The presence of rheumatoid factor (RF) and antinuclear antibodies (ANA) was simultaneously assessed. The results show that MCP-1 serum levels were higher in the healthy aged and lowest in the young, while RANTES increased exclusively in centenarians. Only centenarians had autoantibodies (ANA and RF). sTNF-RI and sTNF-RII were significantly elevated in healthy old subjects compared to the young, and even higher in selected centenarians compared to the other age groups. sCD30 serum levels were significantly raised in centenarians compared to the young, despite absence of circulating CD30+ cells in the peripheral blood of the whole study population. No relationship among serum values of these different members of the TNF-R family was found, despite a strong correlation for sTNF-RI and sTNF-RII in all groups. We hypothesize that the increased chemokine levels in aged people, and raised sCD30 levels in centenarians, may reflect a general shift towards type 0/2 cytokines in normal aging, which may be responsible, at least in part, for the appearance of circulating autoantibodies without definite clinical consequences at advanced age.

Relative Increase of T Cells Expressing the Gamma/Delta Rather Than the Alpha/Beta Receptor in Ataxia-Telangiectasia

In ataxia-telangiectasia, B-cell and T-cell deficiencies are thought to be due to a defect of rearrangements of immunoglobulin and T-cell receptor genes. T cells recognize antigens through two types of CD3-associated receptors: alpha/beta chains on mature cells and gamma/delta chains mostly on immature cells. We studied 10 patients with ataxia-telangiectasia and found that most had a relative increase of circulating T cells bearing gamma/delta receptors rather than alpha/beta receptors, as compared with normal subjects (P less than 0.001). Patients with other immune deficits, including eight with common variable immunodeficiency, one with Wiskott-Aldrich syndrome, two with hyperimmunoglobulinemia E syndrome, and one with severe combined immunodeficiency, had normal ratios of gamma/delta-bearing to alpha/beta-bearing cells. A marked predominance of gamma/delta-bearing T cells was found in a patient with a primary T-cell defect. The relative increase in gamma/delta-bearing T cells in the patients with ataxia-telangiectasia was largely accounted for by cells that reacted with the monoclonal antibody BB3, an apparently distinct subset of T cells that selectively express the C gamma 1 gene product of the T-cell receptor. Although they had normal ratios of gamma/delta-bearing to alpha/beta-bearing T cells, the patients with common variable immunodeficiency had a significant increase (P = 0.01) in the number of T cells expressing C gamma 2 that reacted with the monoclonal antibody delta-TCS-1. We conclude that the increased ratio of gamma/delta-bearing to alpha/beta-bearing T cells in ataxia-telangiectasia may reflect both a recombinational defect that interferes with T-cell and B-cell gene rearrangements and an inability to repair damage to the DNA.

Humoral immunity in aging.

The interactions between B and T lymphocytes, leading to the development of humoral responses, are reviewed with references to the changes occurring in aged people. Aging is perceived as a process of impairment of immune functions; it is known that T cells from aged subjects have a reduced ability to produce IL- 2. However, other functions seem to be upregulated in elderly subjects; indeed, IL- 1, IL- 3, IL- 4, IL- 6 and TNFα production are increased both in aged mice and humans. These cytokines are known to control B cell differentiation, through isotype switch and Ig production. A significant increase in IgG subclasses and IgA is observed in sera of aged subjects. This contrasts with the significant decrease in circulating B lymphocytes. The impairment of primary responses to immunization, and other aspects of humoral immunity, including mucosal responses, autoantibody production and correlations with phenotypic markers of T and B cell subsets, are discussed. (Aging Clin. Exp. Res. 6: 143–150, 1994)

Expression profile of the embryonic markers nanog, OCT-4, SSEA-1, SSEA-4, and frizzled-9 receptor in human periodontal ligament mesenchymal stem cells

Mesenchymal stem cells (MSCs) are self‐renewing cells with the ability to differentiate into various mesodermal‐derived tissues. Recently, we have identified in adult human periodontal ligament (PDL) a population of stem cells (PDL‐MSCs) with the ability to differentiate into osteoblasts and adipocytes. The aim of the present work was to further characterize this population and the expression profile of its cells. To achieve our objective we have used flow cytometry, magnetic cell sorting, cytokine antibody array, and light and electron microscope immunostaining. Our results show that the PDL‐MSCs contain a subpopulation of frizzled‐9 (CD349) positive cells expressing a panel of key mesenchymal and embryonic markers including CD10, CD26, CD29, CD44, CD73, CD90, CD105, CD166, SSEA‐1, and SSEA‐4. They are additionally positive for nanog and Oct‐4; two critical transcription factors directing self‐renewal and pluripotency of embryonic stem cells, and they also express the cytokines EGF and IP‐10. The presence of nanog, Oct‐4, SSEA‐1, and SSEA‐4 suggests that PDL‐MSCs are less differentiated than bone marrow‐derived MSCs. Taken together, these data indicate the presence of immature MSCs in PDL and suggest that the frizzled‐9/Wnt pathway plays an important role in regulating proliferation and differentiation of these cells. J. Cell. Physiol. 225: 123–131, 2010.