Belimumab restores Treg/Th17 balance in patients with refractory systemic lupus erythematosus.

Belimumab, a specific inhibitor of the soluble B lymphocyte stimulator (BlyS), is the first biological drug approved by the United States Food and Drug Administration for the treatment of patients with active systemic lupus erythematosus (SLE) refractory to standard therapy. Given that an imbalance between regulatory T cells (Treg) and interleukin (IL)-17A-secreting T cells (Th17) has been reported in various autoimmune disorders, we assessed the frequency of both Treg and Th17 peripheral blood populations before and after belimumab administration in 20 patients with active SLE refractory to standard therapy. After six months of treatment, the mean SELENA-SLEDAI score as well as the mean anti-double-stranded DNA antibody titers were significantly decreased. In addition, we observed a significant increase in Treg percentages and a parallel, significant decrease in Th17 percentages, accompanied by significantly reduced serum levels of IL-21. In vitro studies showed that Treg purified from belimumab-treated patients were fully functional and displayed a suppressor function similar to that of Treg purified from healthy donors. Belimumab can restore Treg/Th17 balance in SLE patients with uncontrolled disease activity, and this results in decreased flare rate and reduced glucocorticoid dosage.

HCV RNA Genomic sequences and HCV-E2 glycoprotein in sural nerve biopsies from HCV-infected patients with peripheral neuropathy.

AIMS: Peripheral neuropathy (PN), the major neurological complication of chronic HCV infection, is frequently associated with mixed cryoglobulinaemia (MC) and small-vessel systemic vasculitis. While humoral and cell-mediated immune mechanisms are suspected to act together in an aberrant immune response that results in peripheral nerve damage, the role of HCV remains largely speculative. The possible demonstration of HCV in peripheral nerve tissue would obviously assume important pathogenic implications. METHODS: We studied sural nerve biopsies from 11 HCV-positive patients with neuropathic symptoms: five with and six without MC. In situ hybridization (ISH) and immunofluorescence studies were carried out to detect genomic and antigenomic HCV RNA sequences and HCV-encoded E2-glycoprotein, respectively. RESULTS: Epineurial vascular deposits of E2-glycoprotein were found in four (80%) MC and in two (33.3%) non-MC patients, respectively. These findings were enhanced by the perivascular deposition of positive-, though not negative-strand replicative RNA, as also found in the nerve extracts of all patients. Mild inflammatory cell infiltrates with no deposits of immunoglobulins and/or complement proteins were revealed around small vessels, without distinct vasculitis changes between MC and non-MC patients. CONCLUSIONS: These results indicate that nerve vascular HCV RNA/E2 deposits associated to perivascular inflammatory infiltrates were similar in chronically HCV-infected patients, regardless of cryoglobulin occurrence. Given the failure to demonstrate HCV productive infection in the examined sural nerve biopsies, nerve damage is likely to result from virus-triggered immune-mediated mechanisms.

Halting pro-survival autophagy by TGFß inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients.

Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients' plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) ß. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II(+) CAFs. TGFß mediates bort-induced autophagy, and its blockade by LY2109761, a selective TßRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.

Activation-induced cytidine deaminase in B cells of hepatits C virus-related cryoglobulinaemic vasculitis.

Immunoglobulin variable region heavy chain (IgVH ) somatic gene diversification is instrumental in the transformation process that characterizes hepatitis C virus (HCV)-related B cell lymphoproliferative disorders. However, the extent to which activation-induced cytidine deaminase (AID), an enzyme essential for IgV gene somatic hypermutation (SHM), is active in cryoglobulinaemic vasculitis (CV) remains unclear. AID mRNA expression in the peripheral blood of 102 chronically hepatitis C virus (HCV)-infected patients (58 with and 44 without CV) and 26 healthy subjects was investigated using real-time reverse transcription-polymerase chain reaction (RT-PCR). The features of activation-induced cytidine deaminase (AID) protein and mRNA transcripts were explored in liver tissue biopsies and portal tracts isolated using laser capture microdissection. In chronically HCV-infected patients, AID mRNA expression was almost threefold higher in those with than in those without CV and sevenfold higher than in healthy subjects (median-fold: 6.68 versus 2.54, P = 0.03 and versus 0.95, P = 0.0003). AID transcript levels were significantly higher in polyclonal than in clonally restricted B cell preparations in either CV or non-CV patients (median-fold, 15.0 versus 2.70, P = 0.009 and 3.46 versus 1.58, P = 0.02, respectively). AID gene expression was found to be related negatively to age and virological parameters. AID protein was found in portal tracts containing inflammatory cells that, in several instances, expressed AID mRNA transcripts. Our data indicate that the aberrant expression of AID may reflect continuous B cell activation and sustained survival signals in HCV-related CV patients.

Bone marrow fibroblasts parallel multiple myeloma progression in patients and mice: in vitro and in vivo studies.

The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-ß, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial-mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.

Four proteins governing overangiogenic endothelial cell phenotype in patients with multiple myeloma are plausible therapeutic targets.

Bone marrow (BM) angiogenesis has an important role in the initiation and progression of multiple myeloma (MM). We looked at novel mechanisms of vessel formation in patients with MM through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with active MM (MMECs) and ECs of patients with monoclonal gammopathy of undetermined significance (MGECs) and of subjects with benign anemia (normal ECs). Four proteins were found overexpressed in MMECs: filamin A, vimentin, α-crystallin B and 14-3-3ζ/δ protein, not yet linked to overangiogenic phenotype. These proteins gave a typical distribution in the BM of MM patients and in MMECs versus MGECs, plausibly according to a different functional state. Their expression was enhanced by vascular endothelial growth factor, fibroblast growth factor 2, hepatocyte growth factor and MM plasma cell conditioned medium in step with enhancement of MMEC angiogenesis. Their silencing RNA knockdown affected critical MMEC angiogenesis-related functions, such as spreading, migration and tubular morphogenesis. A gradual stabilization of 14-3-3ζ/δ protein was observed, with transition from normal ECs to MGECs and MMECs that may be a critical step for the angiogenic switch in MMECs and maintenance of the cell overangiogenic phenotype. These proteins were substantially impacted by anti-MM drugs, such as bortezomib, lenalidomide and panobinostat. Results suggest that these four proteins could be new targets for the antiangiogenic management of MM patients.

Cyclosporin-A efficacy in chronic idiopathic urticaria.

Common drugs in the therapy of chronic idiopathic urticaria (CIU) include antihistamines alone or combined with corticosteroids, but severe unresponsive patients require alternative treatments. This retrospective study aims to evaluate clinical response and safety of low-dose and long-term oral Cyclosporin-A (CyA) in unresponsive patients. One hundred and ten CIU patients, unresponsive to a previous treatment (antihistamines plus prednisone 0.2 mg/kg/day), received additional oral CyA 1–3 mg/kg/day for 6 months. The patients were subdivided into three groups (A, B, C) according to the different CyA doses. Parameters of clinical efficacy including pruritus, and size and number of wheals were evaluated at baseline, after three and six months. All adverse events were recorded. The mean total symptom severity score decreased by 63% in Group A, 76% in Group B, and 85% in Group C after 6 months. Total disappearance of the symptoms was recorded in 43 patients (39.1%): 7 (28%) of Group A; 12 (37.5%) of Group B and 24 (45%) of Group C. After a mean of 2 months from CyA suspension, 14 patients (11%) had recurrence of symptoms. Minor side effects were noted in 8 patients (7%). Our study indicates that low-dose, long-term CyA therapy is efficacious and safe in severe unresponsive CIU.

Angiogenesis and progression in human melanoma.

In tumor growth, angiogenesis, the process of new-formation of blood vessels from pre-existing ones, is uncontrolled and unlimited in time. The vascular phase is characterized by the new-formation of vascular channels that enhances tumor cell proliferation, local invasion and hematogenous metastasis. Human malignant melanoma is a highly metastatic tumor with poor prognosis, and high resistance to treatment. Parallel with progression, melanoma acquires a rich vascular network, whereas an increasing number of tumor cells express the laminin receptor, which enables their adhesion to the vascular wall, favouring tumor cell extravasation and metastases. Melanoma neovascularization has been correlated with poor prognosis, overall survival, ulceration and increased rate of relapse. Secretion of various angiogenic cytokines, i.e. VEGF-A, FGF-2, PGF-1 and -2, IL-8, and TGF-1 by melanoma cells promote the angiogenic switch and has been correlated to transition from the radial to the vertical growth phase, and to the metastatic phase. Moreover, melanoma cells overexpress alphavbeta3, alphavbeta5, alpha2beta1 and alpha5beta1 integrins and release, together with stromal cells, higher amount of metalloproteases that increasing their invasive potential and angiogenesis. Basing on these observations, different molecular targets of antiangiogenic molecules has be recognized and various antiangiogenic agents are currently in preclinical and clinical trials for melanoma.

CD20-depleting therapy in autoimmune diseases: from basic research to the clinic.

The B lymphocyte-associated antigen CD20 is becoming an important immunotherapy target for autoimmune diseases, although its biological function has not been defined. Besides rheumatoid arthritis, growing experience with B cell-depleting therapy indicates that it may be effective in Sjögren's syndrome, dermatomyositis-polymyositis, systemic lupus erythematosus and some types of vasculitides. However, controlled clinical trials are still lacking for some of these indications. Infection has not been seen as a major limitation to this therapy, but reports of progressive multifocal leukoencephalopathy in an extremely small number of patients are of concern. Here, we review the therapeutic actions of anti-CD20 antibodies, and the recent and ongoing clinical trials with CD20-depleting therapy in autoimmune diseases.

Comparison between filgrastim and lenograstim plus chemotherapy for mobilization of PBPCs.

Recombinant human (rHu) G-CSF has been widely used to treat neutropenia and mobilize PBPCs for their autologous and allogeneic transplantation. It shortens neutropenia and thus reduces the frequency of neutropenic fever. We compared the efficiency of glycosylated rHu and non-glycosylated Hu G-CSF in mobilizing hematopoietic progenitor cells (HPCs). In total, 86 patients were consecutively enrolled for mobilization with CY plus either glycosylated or non-glycosylated G-CSF, and underwent leukapheresis. The HPC content of each collection, toxicity, days of leukapheresis needed to reach the minimum HPC target and days to recover WBC (> or =500 and >1000/mm(3)) and plts (>50 000/mm(3)) were evaluated. Glycosylated G-CSF mobilized more CD34+ cells than did the non-glycosylated form. The ability to reach a collection target of >3 x 10(6) CD34+/kg body weight in two leukaphereses was higher for glycosylated G-CSF. No significant differences between the two regimens were observed with regard to toxicity and days to WBC and plt recovery. High-dose CY plus glycosylated G-CSF achieved adequate mobilization and the collection target more quickly and with fewer leukaphereses.

Managing myelodysplastic symptoms in elderly patients.

Most patients with myelodysplastic syndromes (MDS) are elderly (median age range 65 to 70 years); as a consequence, the incidence and prevalence of these diseases are rising as the population ages. Physicians are often uncertain about how to identify patients who may benefit from specific treatment strategies. The International Prognostic Scoring System is a widely used tool to assess the risk of transformation to leukemia and to guide treatment decisions, but it fails to take into account many aspects of treating elderly patients, including comorbid illnesses, secondary causes of MDS, prior therapy for MDS, and other age-related health, functional, cognitive, and social problems that affect the outcome and managing of myelodysplastic symptoms. Patients with low-risk disease traditionally have been given only best supportive care, but evidence is increasing that treatment with novel non-conventional drugs such as lenalidomide or methyltransferase inhibitors may influence the natural history of the disease and should be used in conjunction with supportive-care measures. Supportive care of these patients could also be improved in order to enhance their quality of life and functional performance. Elderly patients commonly have multiple medical problems and use medications to deal with these. In addition, they are more likely to have more than one health care provider. These factors all increase the risk of drug interactions and the consequent treatment of toxicities. Manifestations of common toxicities or illnesses may be more subtle in the elderly, owing to age-associated functional deficits in multiple organ systems. Particularly important to the elderly MDS patient is the age-related decline in normal bone marrow function, including the diminished capacity of response to stressors such as infection or myelosuppressive treatments. Through the integration of geriatric and oncological strategies, a personalized approach toward this unique population may be applied. As with many diseases in the elderly, reliance on family members or friends to maintain the prescribed treatments, including travel to and from appointments, may place additional stressors on the patient and his/her support network. Careful evaluation and knowledge of functional status, ability to tolerate treatments, effect of disease progression, and general overall health conditions can provide the best opportunity to support these patients. Immediate assessment of daily living activities may detect deficiencies or deficits that often require early interventions.

[The advent of biosimilars: new rules to guarantee patient safety]. / L'avvento dei biosimili: nuove regole per garantire la sicurezza del paziente.

Biosimilars are medicinal products proposed as copies of biotech drugs whose patents have expired. Their entry into the therapeutic armamentarium entails knowledge of the rules established in Europe relevant to their use in clinical practice. In September 2007, an Italian panel of experts comprising two nephrologists, a clinical immunologist, an oncohematologist, a pharmacologist, and a hospital pharmacist examined the main features of biotech drugs and the issues faced by the regulatory authorities in the definition of a specific approval pathway for biosimilars in Europe. The panel of experts agreed that it is important to inform the medical and scientific community that biosimilars are not exact copies of their reference products; therefore the rules governing their clinical use are not the same as those established for biotech drugs in general. Patient safety should be the fundamental principle guiding therapeutic choices, and making these choices should be the prerogative of physicians.

Delayed complete remission in a patient with multiple myeloma.

We report a strikingly positive, late response to bortezomib in conjunction with pegylated liposomal doxorubicin in a 79-year old woman with multiple myeloma (MM). The patient obtained a partial remission after eight courses of therapy and a complete remission about 10 months after the end of therapy. This delayed complete remission may be similar to the spontaneous regression reported for other malignancies such as melanoma or lymphoma. We postulate that the immune response and a persistent anti-angiogenic effect of bortezomib could well explain the delayed complete remission in our patient.

Overexpression of interleukin-12 and T helper 1 predominance in lupus nephritis.

Imbalance of cytokine homeostasis is a prominent feature of both experimental and human systemic lupus erythematosus (SLE). Because interleukin (IL)-12 promotes interferon (IFN)-gamma production leading to polarization of peripheral cells toward a T helper (Th) 1 phenotype, we investigated its role in lupus nephritis (LN). Soluble Th1 and Th2 cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in sera and urines of SLE patients and controls. Th1/Th2 peripheral lymphocyte polarization was determined by flow cytometry. Glomerular accumulation of IL-12 was evaluated by immunohistochemistry, whereas urinary IL-12 was evaluated by ELISA. Higher serum IL-12 levels in SLE were associated with LN, whereas IL-4 was unrelated to the renal damage. Peripheral cells from LN patients showed a Th1 phenotype with a high IFN-gamma expression that paralleled the severity of renal damage. IL-12 was present within glomerular mononuclear cells in classes IV and V LN, and its accumulation was correlated strongly with urinary levels. IL-12 overexpression in SLE may contribute to the development of LN. Both serum and urinary IL-12 elevation reflect its glomerular production and parallel Th1 polarization of peripheral T cells and high IFN-gamma production. In SLE patients, IL-12 measurement may thus be predictive of the development of LN.

Double-filtration plasmapheresis in the treatment of leg ulcers in cryoglobulinemia.

Hepatitis C virus (HCV) is the major cause of cryoglobulinemia. Skin lesions are frequent and can be cured from the removal of cryoglobulins by therapeutic apheresis. We describe a case of HCV-positive type I cryoglobulinemia with severe leg ulcers, not responsive to antiviral and immunosuppressive treatment. Thirty sessions of double filtration plasmapheresis were performed, over a period of 6 months, with no other associated treatment. Before and after each session an assessment of immunoglobulins, complement, cryocrit, and fibrinogen was made. HCV RNA levels were determined in serum cryoprecipitate, supernatant before and after each session, and in the collection bag. No differences in pre and postapheresis values were observed in the serum concentrations and the supernatant, whereas the postapheresis cryoprecipitate showed a significantly reduced viral load (P < 0.02) as compared with the preapheresis values. There was improvement in the condition of ulcers in the leg during apheresis and had completely regressed by the end of the cycle.

Current and emerging therapeutic approaches in HCV-related mixed cryoglobulinemia.

Recognition of hepatitis C virus (HCV) as an etiological factor in mixed cryoglobulinemia (MC) has dramatically changed our point of view in its treatment. Emphasis is placed on abatement and clearance of viral load and deletion of clonal expansions of IgM molecules with rheumatoid factor activity-synthesising B cells. The purpose of this review is to discuss the underlying scientific rationale and results of clinical studies of new treatment approaches to MC, with a focus on cell-depleting therapies and chemokine blockade. Additional antiviral agents directed to several phases of HCV life cycle acting with different or alternate mechanisms are proposed with the goal to enhance response rates more broadly suitable for MC patients with vasculitis and peripheral neuropathies. The majority of the available data on these new treatment approaches stems from open-label studies, but controlled trials are under way. Therapy directed against chemokines and/or cytokines represents an interesting and promising future target.

Vasculogenic mimicry by bone marrow macrophages in patients with multiple myeloma.

Bone marrow macrophages of patients with active and nonactive multiple myeloma (MM), monoclonal gammopathies of undetermined significance (MGUS) and benign anemia (controls) were stimulated for 7 days with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and analysed for the expression of endothelial cell (EC) markers by reverse transcription (RT)-PCR, real-time RT-PCR, western blot and immunofluorescence. Their vasculogenic ability was investigated in vitro in a Matrigel assay and in vivo on bone marrow biopsies through dual immunofluorescence and confocal laser microscopy. Active MM macrophages exposed to VEGF and bFGF acquired EC markers and formed capillary-like structures mimicking paired bone marrow ECs (multiple myeloma patient-derived endothelial cells, MMECs), with major responsiveness compared to macrophages from nonactive MM, MGUS or controls. Bone marrow biopsies of active MM harbored 'mosaic' vessels, being formed by MMECs, EC-like macrophages and macrophages themselves. These figures were rare in nonactive MM and absent in MGUS or controls. Our data indicate that macrophages contribute to build neovessels in active MM through vasculogenic mimicry, and this ability proceeds parallel to progression of the plasma cell tumors. Macrophages may be a target for the MM antivascular treatment.

Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus.

The ability of the immune system to distinguish between self and non-self is critical to the functioning of the immune response. A breakdown in these mechanisms can lead to the onset of autoimmune disease. Clinical and molecular data suggest that shared immunogenetic mechanisms lead to the autoimmune process. The most studied part of the autoimmune process is the human leukocyte antigen (HLA) region. Recently, progress has been made in narrowing down HLA cluster classifications based on structural and functional features of HLA alleles. Using this approach we have investigated 175 patients with hepatitis C virus (HCV)-induced type II cryoglobulinemia (MC), and compared them to a control group of 14,923 bone marrow donors. Additionally, we investigated the frequency of HLA homozygosity in the same groups of subjects. Our results provide evidence of a role for DR5 and DQ3 HLA class II clusters and a higher frequency of HLA homozygous leading to the clinical outcome of type II mixed cryoglobulinemic autoimmune disease. The DR5 cluster is characterized by a Glu in beta 9 and its polymorphism is connected with preferred anchors at beta 9 of the binding peptide, while the DQ3 cluster is characterized by Glu B86 and Leu B87, which allows the binding of large hydrophobic amino acids at p1 of the binding peptide. The mechanisms by which variations in HLA lead to autoimmunity remain unknown, although they are likely to be mediated by continuous presentation of HCV epitopes to T cells and a genetic background that limits the effective clearance of HCV. The results presented in this paper have increased our knowledge of the mechanism of autoimmune disease and B-cell lymphoproliferation during HCV infection. The work was performed in accordance with the principles of the 1983 Declaration of Helsinki. There is no conflict of interest.

New therapies in multiple myeloma.

The melphalan-prednisone regimen has been considered as standard therapy for patients with multiple myeloma (MM) for many years. Recently, high-dose chemotherapy with stem-cell support has extended progression-free survival and increased overall survival, and it is now considered conventional therapy in younger patients. However, most patients relapse and the salvage treatment is not very effective. New active drugs, including immunomodulatory agents, thalidomide (Thal) and lenalidomide, and the proteasome inhibitor bortezomib, have shown promising anti-myeloma activity. These novel treatments are aimed at overcoming resistance of tumour cells to conventional chemotherapy, acting both directly on myeloma cells and indirectly by blocking the interactions of myeloma cells with their local microenvironment and suppressing growth and survival signals induced by autocrine and paracrine loops in the bone marrow. Thal has been widely studied, mostly in combination regimens in patients with relapsed MM and, more recently, in front-line therapy, showing efficacy in terms of response rate and event-free survival. Bortezomib has been found to possess remarkable activity, especially in combination with other chemotherapeutic agents, in relapsed/refractory and newly diagnosed MM, as well as in patients presenting adverse prognostic factors. Lenalidomide, in combination with dexamethasone, is showing high overall response rates in relapsed and refractory MM and promising results also in first-line therapy. In this paper, the results of the most significant trials with Thal, bortezomib and lenalidomide are reported. Several ongoing clinical studies will hopefully allow the identification of the most active combinations capable of improving survival in patients with MM.