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Background: All implant compounds undergo an electrochemical process when in contact with biological fluids, as well as mechanical corrosion due to abrasive wear, with production of metal debris that may inhibit repair processes. None of the commonly-used methods can diagnose implant allergies when used singly, therefore a panel of tests should be performed on allergic patients as pre-operative screening, or when a postoperative metal sensitisation is suspected., Methods: We analysed patients with painful prostheses and subjects prone to allergies using the Patch Test in comparison with the Lymphocyte Transformation Test. Cytokine production was evaluated to identify prognostic markers for early diagnosis of aseptic loosening. Metal debris endocytosis and cytoskeletal rearrangement was visualised by confocal microscopy., Results: Our results demonstrate that the Lymphocyte Transformation Test can identify patients who have a predisposition to develop allergic reactions and can confirm the diagnosis of hypersensitivity in patients with painful prostheses. The prevalence of a Th2-cytokine pattern may be used to identify predisposition to the development of allergic diseases, while the selective presence of osteoclastogenic cytokines may be used as predictor of a negative outcome in patients with painful prosthesis. The hypothesis of the prognostic value of these cytokines as early markers of aseptic loosening is attractive, but its confirmation would require extensive testing., Conclusions: The Lymphocyte Transformation Test is the most suitable method for testing systemic allergies. We suggest that the combined use of the Patch Test and the Lymphocyte Transformation Test, associated with cytokine detection in selected patients, could provide a useful tool for preventive evaluation of immune reactivity in patients undergoing primary joint replacement surgery, and for clinical monitoring of the possible onset of a metal sensitization in patients with implanted devices.

Purpose: We evaluated the risk of hypersensitivity to metals in a population of consecutive subjects undergoing a total knee arthroplasty (TKA). We also proposed a diagnostic pathway to address any sensitivity to metals. We finally presented the mid-term outcomes of a full non allergenic knee implant., Methods: We developed a protocol based on the medical history, patch testing, and on specific laboratory assays, in order to assess a sensitization to metals. Twenty-four patients (25 knees) with referred or suspected allergy to metals were found in more than 1,000 treated patients, with a mean age of 72.9 years. We proceeded to a radiologic study, a clinical evaluation by the visual analogic scale (VAS), and Knee Society rating system (KSS). In all cases a full anallergic cemented implant with an oxidized zirconium femoral component and an all-polyethylene tibial baseplate was chosen., Results: Four (16.6%) of the 24 patients were considered to be hypersensitive to metals. The mean follow-up was 79.2 months. No patient reported any reaction related to hypersensitivity or complications after TKA. The VAS improved from a mean preoperative value of 7.2 to 1.8 postoperatively; the KSS and the functional score increased from 38 to 91 points and from 39 to 88 points, respectively., Conclusions: We consider careful research of medical history for metals hypersensitivity crucial, and we perform patch testing and lab assays in case of doubtful sensitization. The choice of a modern hypoallergenic implant may prevent any kind of potential reactions.

Background: Symptomatic severe osteoarthritis and hip osteoporotic fractures are the main conditions requiring total hip arthroplasty (THA), whereas total knee arthroplasty (TKA) is mainly performed for pain, disability or deformity due to osteoarthritis. After surgery, some patients suffer from "painful prosthesis", which currently represents a clinical problem., Methods: A systematic review of scientific literature has been performed. A panel of experts has examined the issue of persistent pain following total hip or knee arthroplasty, in order to characterize etiopathological mechanisms and define how to cope with this condition., Results: Four major categories (non infective, septic, other and idiopathic causes) have been identified as possible origin of persistent pain after total joint arthroplasty (TJA). Time to surgery, pain level and function impairment before surgical intervention, mechanical stress following prosthesis implant, osseointegration deficiency, and post-traumatic or allergic inflammatory response are all factors playing an important role in causing persistent pain after joint arthroplasty. Diagnosis of persistent pain should be made in case of post-operative pain (self-reported as VAS ≥3) persisting for at least 4 months after surgery, or new onset of pain (VAS ≥3) after the first 4 months, lasting ≥2 months. Acute pain reported as VAS score ≥7 in patients who underwent TJA should be always immediately investigated., Conclusions: The cause of pain needs always to be indentified and removed whenever possible. Implant revision is indicated only when septic or aseptic loosening is diagnosed. Current evidence has shown that peri-and/or post-operative administration of bisphosphonates may have a role in pain management and periprosthetic bone loss prevention.

Superoxide anion (O(2) (•-)) is overproduced in joint inflammation, rheumatoid arthritis, and osteoarthritis. Increased O(2) (•-) production leads to tissue damage, articular degeneration, and pain. In these conditions, the physiological defense against O(2) (•-), superoxide dismutases (SOD) are decreased. The Mn(II) complex MnL4 is a potent SOD mimetic, and in this study it was tested in inflammatory and osteoarticular rat pain models. In vivo protocols were approved by the animal Ethical Committee of the University of Florence. Pain was measured by paw pressure and hind limb weight bearing alterations tests. MnL4 (15 mg kg(-1)) acutely administered, significantly reduced pain induced by carrageenan, complete Freund's adjuvant (CFA), and sodium monoiodoacetate (MIA). In CFA and MIA protocols, it ameliorated the alteration of postural equilibrium. When administered by osmotic pump in the MIA osteoarthritis, MnL4 reduced pain, articular derangement, plasma TNF alpha levels, and protein carbonylation. The scaffold ring was ineffective. MnL4 (10(-7) M) prevented the lipid peroxidation of isolated human chondrocytes when O(2) (•-) was produced by RAW 264.7. MnL4 behaves as a potent pain reliever in acute inflammatory and chronic articular pain, being its efficacy related to antioxidant property. Therefore MnL4 appears as a novel protective compound potentially suitable for the treatment of joint diseases.

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.

Within the past several years, the relation between diet and health has been accepted by the mainstream nutrition community and in this connection interest in the physiological role of bioactive compounds present in plants has dramatically increased over the last decade.The phytoestrogens are bioactive molecules present as nutritional constituents of widely consumed vegetables. Their name derives from the fact that they are able to bind to estrogen receptors and to induce an estrogenic/antiestrogenic response in target tissues. Natural estrogens are involved in a multiplicity of programmed events in target tissues as uterus, breast, pituitary gland and hormone responsive tumors. Phytoestrogens are present in many human foodstuffs including fruits (plum, pear, apple grape berries, …), vegetables (beans, sprouts, cabbage, spinach, soybeans, grains, hops, garlic, onion,…), wine, tea, and they have been identified in a number of botanical dietary supplements. They include a wide variety of structurally different compounds such as isoflavones, mainly found in soy, lignans found in grains, stilbenes found in the skin of grapes. Other less investigated compounds include flavones, flavans, isoflavanes and coumestans. The estrogenic or antiestrogenic activity of any chemicals depends on the ability of the compound to interact with the ERs (ERα , ERβ ).This article reported the knowledge about the activity of phytoestrogens from a pharmacological point of view for their estrogenicity or antiestrogenicity.

Background. Clodronate is used in high bone resorption diseases. Its action was defined as "cytotoxic" based on the induced cellular ATP loss, without any experimental verification of reversibility. In the present report the reversibility of clodronate action was tested on cultured human osteoclastic cell cultures. As "in vitro" bioeffects of clodronate are reversible, this compound should not be defined as "cytotoxic".Introduction. Bisphosphonates are pyrophosphate analogs able to inhibit osteoclast-mediated bone resorption widely used in the treatment of diseases with high bone turnover. Several evidences have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2)side chain. The nitrogen-containing bisphosphonates act on osteoclasts by preventing protein prenylation, while non-nitrogen-containing bisphosphonates, like clodronate, are metabolized intracellularly to a β-γ-methylene analog of ATP that induces inhibition of the ADP/ATP translocase.Materials and Methods. In order to evaluate clodronate effects on osteoclastic cells and the bioreversibility of its action, we have used a human preosteoclastic (FLG 29.1) cell line and primary cultures of human osteoclast-like (HOC) cells. Functional and differentiative modifications were evaluated with immunocytochemical tartrate-resistant acid phosphatase activity (TRAcP) assay and with rapid quantitative detection of the complex "matrix metalloproteinase 9/tissue inhibitor of metalloproteinase" (MMP9/TIMP1) by RT-PCR analysis based on "TaqMan" technology. The apoptosis phenomenon were detected by DNA ladder analysis and quantified by counting apoptotic cells with Transmission Electron Microscopy (TEM) analysis. Adenosine-5'-[ β - γ -dichloromethylene] triphosphate (AppCCl(2)p) was detected and identified in cell extract by HPLC-ESI-MS-MS Mass Spectrometry. Intracellular ATP modulation in the presence of clodronate was evaluated by luciferin-luciferase assay. The Mann-Whitney "U" test was conducted for statistical analysis.Results. We found that clodronate inhibited both proliferation and differentiative features of cells of the osteoclastic lineage. Furthermore, treatment of both cell types with clodronate caused apoptosis, generation of measurable levels of AppCCl(2)p, and reduction of intracellular ATP levels. Addition of ATP to the culture medium caused an inhibition of the biological actions of clodronate on the human osteoclastic cell lineage.Conclusions. These data indicate that intracellular accumulation of the metabolite AppCCl(2)p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP.

The pattern of cytokine production of skin-infiltrating T cells from patients with progressive systemic sclerosis was investigated. Most CD4+ T-cell clones generated from skin biopsy specimens showed a type 2 helper (Th2) cytokine profile (production of interleukin-4, but no interferon (IFN)-gamma). High interleukin-4 but little or no IFN-gamma mRNA expression was found by in situ hybridization in skin perivascular mononuclear cell infiltrates. The immunohistochemical analysis revealed CD30 expression by high numbers of CD4+ T cells in the same specimens. Finally, the great majority of patients with diffuse disease had elevated levels of soluble CD30 in their sera. These data suggest the existence in patients with progressive systemic sclerosis of a predominant activation of Th2-like T cells, which may account for the major alterations (endothelial cell injury, fibrosis, and autoantibody production) occurring in this disease.

Systemic sclerosis (scleroderma) is characterized by excessive deposition of extracellular matrix constituents. Although it has been proposed that tissue fibrosis is due to increased fibroblast synthesis of various collagen polypeptides, there is some experimental evidence that patients with systemic sclerosis have a defect in the control of fibroblast growth. The myb family of genes includes, among others, the c-myb proto-oncogene and the structurally related gene, B-myb, which are both implicated in the regulation of differentiation and/or proliferation of hematopoietic and nonhematopoietic cells. To elucidate the molecular basis responsible for scleroderma fibroblast proliferation, we therefore elected to investigate the expression of c-myb and B-myb genes in scleroderma and control cells. Using the reverse transcriptase polymerase chain reaction technique, we detected c-myb transcripts in scleroderma skin fibroblasts rendered quiescent by serum deprivation. Under the same experimental conditions, c-myb message was not found in normal skin fibroblasts, but, after serum stimulation, c-myb RNA was clearly evident from 3 to 72 h in both normal and pathologic cells. Treatment of these cells with c-myb antisense oligonucleotides caused downregulation of c-myb expression, and the inhibition of scleroderma fibroblast proliferation was 42%, whereas in normal fibroblasts the inhibition was weaker (22%). In contrast to c-myb, in normal and scleroderma fibroblasts the level of expression of B-myb correlated with cell proliferation assessed by cell count, and densitometric analysis showed that B-myb message was 1.5-5 times higher in most of pathologic cells studied. The antisense B-myb oligonucleotides had a weaker antiproliferative effect compared with antisense c-myb, inhibiting scleroderma and normal fibroblasts by 23% and 13%, respectively. These data suggest that the B-myb and c-myb genes may play a role in scleroderma fibroblast proliferation and function.

The CD40 antigen, a member of the nerve growth factor/tumor necrosis factor receptor family, is expressed on all mature B lymphocytes and plays a crucial role in B cell activation, T cell-dependent antigen-driven isotype switching and germinal center formation. We have analyzed CD40 expression and function during mouse B cell development by examining B cell precursors in normal mice and in transgenic animals in which B cell development is frozen at discrete stages. These models included RAG-2-/- mice, and transgenic littermates that express a mu heavy chain and/or the bcl-2 proto-oncogene transgene. CD40 was undetectable at the pro-B cell stage, but was expressed, although at low levels, on pre-B cells. However, pre-B cells failed to respond to CD40 triggering either by expression of CD23 or by proliferation in the presence of IL-4. Overexpression of bcl-2 increased the density of CD40 expression on pre-B cells: these cells respond to CD40 ligation by expressing CD23 and by proliferating in the presence of IL-4.

The specific immune response against the malignant cells was investigated in patients with urinary bladder or larynx cancer. Lymphocytes from lymph nodes that drain the tumor site were tested for their proliferative and cytotoxic capacities against autologous malignant cells isolated from the primary tumor. In no occasion was a proliferative or a cytotoxic response observed. However, when the lymph node cell suspensions were depleted of cells expressing both OKM1 and Leu-7 markers by rosetting with the appropriate mAbs, a proliferative response could be observed. The lymphocytes responded to autologous tumor cells only if IL-2 was added to the cultures. IL-2 alone induced some cell proliferation, which was not, however, comparable to that observed in response to both IL-2 and tumor cells. A panel of allogeneic tumor cells consistently failed to stimulate OKM1-, Leu-7- cells in vitro. Response to autologous tumor cells was not caused by HLA-encoded molecules, as occurs in the autologous mixed lymphocyte reaction, since OKM1-, Leu-7- cells failed to be stimulated by autologous non-T cells. A proliferative response was observed only with cells from lymph nodes that had been classified as invaded by malignant cells according to histopathologic criteria. Cells from noninvaded lymph nodes consistently failed to respond. Cells stimulated with autologous tumor cells could be expanded in short-term lines by continuous addition of IL-2 and malignant cells. One of these lines, which comprised mainly T8+ cells, was stimulated to proliferate only by autologous tumor cells, and its proliferative response was inhibitable by anti-class I and not by anti-class II mAbs. This line showed lytic capacities against autologous malignant targets, while it was inefficient against all of the other allogeneic cells tested. In another set of experiments, the mechanisms whereby exogenous IL-2 had to be added to the cultures to sustain a proliferative response against neoplastic cells were investigated. When cocultured with autologous malignant cells, OKM1-, Leu-7- lymphocytes expressed IL-2 receptors, as could be assessed by anti-Tac fluorescent staining. Under these culture conditions, these cells did not produce IL-2, and no proliferation was observed. Addition of purified IL-1 to the cultures induced IL-2 production and cell proliferation. It is concluded that metastatic lymph nodes contain a T cell population that can be detected in a proliferative assay when both suppressor cells are removed and the appropriate molecular signals are supplied.