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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 cellmediated 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. [ABSTRACT FROM AUTHOR]

Perosa F, Prete M, Racanelli V, Dammacco F (University of Bari Medical School, Bari, Italy). CD20-depleting therapy in autoimmune diseases: from basic research to the clinic (Review). J Intern Med 2010; 267: 260–277. 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. [ABSTRACT FROM AUTHOR]

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. J. Clin. Apheresis, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Aims: To correlate microvascular density and erythropoietin (Epo)/Epo-receptor (EpoR) expression in endothelial and tumour cells with histopathological type in hepatocellular carcinoma (HCC). Methods and results: Specimens of primary HCC obtained from 50 patients who had undergone curative hepatectomy were investigated immunohistochemically by using anti-CD31, anti-Epo and anti-EpoR antibodies. Poorly differentiated HCC had a higher degree of vascularization than other stages and Epo/EpoR expression in both tumour and endothelial cells increased in parallel with grade of malignancy and was highly correlated with the extent of angiogenesis. Conclusions: Epo/EpoR levels correlate with angiogenesis and progression of patients with HCC and these findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by hepatic tumour cells and it affects vascular endothelial cells via its receptors and promotes angiogenesis in a paracrine manner. It is thus suggested that Epo is an important factor in hepatic tumour angiogenesis. Understanding the mechanisms of HCC angiogenesis provides a basis for a rational approach to the development of antiangiogenic therapy in patients with hepatic cancer. [ABSTRACT FROM AUTHOR]

Immunohistochemistry provides valuable information concerning the localization and distribution of hepatitis C virus (HCV)-related proteins in histological sections of liver tissue, but does not readily permit their quantitation in individual cells and the staining intensity of cell immunodeposits cannot be calibrated with the current number of antigen molecules. We specifically detected and quantitated HCV core protein in single hepatocytes by coupling laser capture microdissection (LCM) with a sensitive enzyme-linked immunosorbent assay (ELISA). Quantitation of HCV core protein per cell was carried out on liver tissue cells obtained by LCM from fixed and stained frozen sections of 10 HCV-positive patients with chronic active hepatitis (CAH). Macromolecules from captured cells were solubilized in an extraction buffer and directly assayed for core protein using a sandwich ELISA. Calibration was achieved by developing a standard curve based on known concentrations of HCV core protein. Precision, linearity and sensitivity were verified for known numbers of microdissected tissue cells. In this study, the concentration of HCV core protein in single hepatocytes ranged from 7 to 56 pg/cell. Specificity was verified on 10 replicates of 10 HCV-negative liver tissues. Immunohistochemical staining of HCV core protein was compared with the results of the soluble immunoassay for the adjacent liver tissue sections. Independent scoring of HCV immunostaining failed to parallel the LCM quantitative immunoassay. LCM-based immunoassay significantly expands our ability to investigate function-related antigens in apparently pure cell populations in HCV infection. [ABSTRACT FROM AUTHOR]

Cryoglobulins are cold-precipitable immunoglobulins associated with a number of infectious, autoimmune and neoplastic disorders. Their appearance along with rheumatoid factor (RF) can be considered a normal event in the clearance of immune complexes and rarely produces any symptoms. The association between hepatitis C virus (HCV) and mixed cryoglobulinemia (MC) has been rendered evident since the recognition of serological markers of HCV infection. There is thus every reason to suppose that direct or indirect involvement of B cells on the part of the HCV results in their persistent stimulation, clonal expansion and release of molecules with RF activity. The formation of RF/IgG immune complexes is the key pathogenetic mechanism. The close correlation between HCV infection and MC also throws new light on the interpretation of autoimmune phenomena in the course of viral infection and on the close link between autoimmune diseases and lymphoproliferative disorders. The higher risk of non-Hodgkin's lymphoma (NHL) displayed by HCV positive subjects, especially in the Mediterranean basin, suggests that the HCV's chronic lymphoproliferative drive may progress towards frank lymphoid neoplasia. The presence of MC does not represent an in situ or ‘occult’ NHL, because recent evidences indicate that none of the clones interpreted as predominant displays the molecular features of a true neoplastic process. The cryoglobulinemic syndrome is probably the consequence of pathogenic noxae that act upon the immune system of a host in which regulation of the peripheral T cell response appears to be in some way altered. [ABSTRACT FROM AUTHOR]

BackgroundMany data suggest that the density of mast cells is highly correlated with the extent of both normal and pathological angiogenesis. ObjectiveIn this study we have compared in an in vivo assay, the chick embryo chorioallantoic membrane, the angiogenic potential of mast cell suspensions isolated from rats, degranulated mast cells and their secretory granules. MethodsGelatin sponges adsorbed with cell suspensions of rat mast cells, degranulated mast cells and their secretory granules were implanted on the top of the chorioallantoic membrane at day 8 of incubation. At day 12 the angiogenic response was evaluated macroscopically, microscopically and by a morphometric method of ‘point counting’. ResultsIsolated mast cells and their secretory granules, but not degranulated mast cells, induced an angiogenic response in the chorioallantoic membrane. The addition of antifibroblast growth factor-2 or antivascular endothelial growth factor antibodies reduced the angiogenic response of both mast cells and their secretory granules by 50% and 30%, respectively. ConclusionThese data support the evidence that the angiogenic properties of mast cells depend on the angiogenic molecules contained in their secretory granules and indicate that fibroblast growth factor-2 and vascular endothelial growth factor are the angiogenic cytokines primarily and perhaps synergistically responsible for this vasoproliferative activity. [ABSTRACT FROM AUTHOR]

The thyroglobulin antibodies present in the sera of thirteen leprosy patients were shown to be exclusively or predominantly macroglobulin in character when studied by diethylaminoethylcellulose chromatography and preparative ultracentrifugation. This was confirmed by immunoelectrophoresis and analytical ultracentrifugation of the fractions containing thyroglobulin antibodies, as well as by tests with 2-mercaptoethanol. The anti-thyroglobulin specificity of the antibodies showing macroglobulin character was ascertained by absorption experiments with human thyroglobulin; it was ruled out by absorption with FII that the 19S character might result from the formation of complexes with anti-γ-globulin factors. The conditions are discussed which may affect the 7S or 19S character of antibodies, in particular the antibody titre, the amount of antigenic stimulation and the vigour of the subject's immune response. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which discusses a case study of patients with Primary Central Nervous system Lymphoma (PCNSL).

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. [ABSTRACT FROM AUTHOR]

Hybridization of a non-secreting mouse myeloma cell line NSO with splenocytes from a BALB/c mouse hyperimmunized with the anti-CD4 monoclonal antibody (mAb) HP2/6 generated the anti-idiotypic (anti-id)mAb F11-2113, F11-2302 and F11-2444, which recognize idiotope(s) (id) that are the same or spatially close to each other and are located outside the antigen-combining site of the immunizing mAb. Binding and inhibition assay showed that id are not expressed either on other mouse anti-CD4 mAb or polyclonal immunoglobulins (Ig). Western blotting analysis showed that the id defined by anti-id mAb F11-2113, F11-2302 and F11-2444 are similarly expressed on separated heavy and light chains of mAb HP2/6 and suggested they are likely to be ‘'sequence-dependent'’ since their expression is conserved following SDS and reducing reagents treatment. This finding is unique inasmuch as ‘'sequence-dependent'’ id similarly expressed on heavy and light chains have been described only on a mouse monoclonal auto-antibody with immunoregulatory properties. [ABSTRACT FROM AUTHOR]

Summary No monoclonal antibodies (mAbs) are presently available to measure the total amount of β2-microglobulin-free HLA class I heavy chain (FHC) in sera. The available ELISA-based double determinant immunoassay (DDIA), established to measure FHC, uses two mAbs (TP25.99 and HC-10) that recognize a monomorphic determinant expressed on all HLA-B/C FHC products and a determinant expressed only on some HLA-A FHC products. This restricted reactivity implies that, in addition to HLA-B/C, HLA-A FHC products are also detected in individuals bearing HLA A3 and/or A30 allotypes. The aim of this study was to establish whether such restriction results in the detection of low FHC levels in individuals lacking HLA A3 and/or A30 allospecificities. The FHC mean concentration (± SD) in 294 healthy blood/bone marrow donors (HBDs) was 0.24 (± 0.2) mg/l. The grouping of HBDs according to their HLA-A FHC product reactivity with one, both or no mAbs did not result in any statistically significant differences (Mann–Whitney test: P > 0.05) between their median FHC concentrations. Since the absence of differences in their FHC levels was not attributable to a difference in the percentage distribution of HLA allotypes associated with high or low HLA-B/C FHC expression, our results indicate that FHC HLA A3 and/or A30 products detected in DDIA by these two mAbs only minimally contribute to FHC serum expression and that the assay is not limited by the failure to detect HLA-A FHC products in A3 - and/or A30 - individuals. [ABSTRACT FROM AUTHOR]

Interleukin 17A (IL17A), a cytokine involved in allergy, inflammation and osteoclastogenesis, was investigated in multiple myeloma (MM) to assess its role in the osteoclast (OC)-like activity of marrow immature dendritic cells (iDCs). Comparing nine MM patients with control subjects affected by monoclonal gammopathy of undetermined significance, we found high IL17A expression in the marrow plasma of MM patients in parallel with its deposits within the stromal matrix. Increased expression of the IL17A receptor (IL17RA) was also found in primary myeloma iDCs, which underwent OC-like transdifferentiation after IL17A stimulation. To assess the role of IL17A, we measured the activity of the IL17/IL17RA pathway in IL17A-transdifferentiated iDCs and the expression of functional OC genes by Western blotting and real-time polymerase chain reaction. These cells showed increased RNA transcription of genes enrolled in the maturation of OCs, while NFATC1 and FOS were induced by IL17A, independently of NFKB1 phosphorylation. Moreover, the concurrent phosphorylation of the Lip isoform of CEBPB and the down-regulation of MAFB supported the activation of IL17RA pathway in OC-like transdifferentiated iDCs that was apparently unrelated to TNFRSF11A signalling. These data emphasize the involvement of iDCs in MM hyperactive osteoclastogenesis and suggest that their bone resorption activity is also regulated, at least in vitro, by IL17RA., (© 2013 John Wiley & Sons Ltd.)

Recently, genetically modified mesenchymal stem cells (MSCs) have been exploited to deliver anti-cancer bio-drugs directly within the tumour mass. Here, we explored whether adipose-derived MSCs (AD-MSCs), engineered to express the pro-apoptotic ligand TRAIL (also known as TNFSF10), kill multiple myeloma (MM) cells and migrate towards MM cells in vitro. Different MM cell lines were assessed for their sensitivity to recombinant human (rh) TRAIL alone and in combination with the proteasome inhibitor bortezomib, which was shown to enhance the effect of rhTRAIL. TRAIL(+) -AD-MSCs were co-cultured with bortezomib-pretreated MM cells and their killing activity was evaluated in presence or absence of caspase inhibition. AD-MSC migration towards media conditioned by both myeloma cells and myeloma bone fragments was also investigated. Despite moderate MM cell sensitivity to rhTRAIL, TRAIL(+) -AD-MSCs in combination with bortezomib significantly induced myeloma cell death. This effect was associated with caspase-8 activation and abrogated by capsase inhibition. On the other hand, co-culture experiments were performed to evaluate whether unmodified AD-MSCs affect myeloma cell growth in vitro. AD-MSCs appeared ineffective on myeloma cell growth and showed migratory capacity towards MM cells in vitro. These data emphasize the anti-myeloma activity of TRAIL-engineered AD-MSCs and provide support for a future model of a cell-based approach against MM., (© 2012 Blackwell Publishing Ltd.)

Randomized clinical studies, carried out in patients with haematological malignancies and with solid tumours, have consistently demonstrated that treatment with recombinant human erythropoietin (Epo) increases haemoglobin levels, reduces blood transfusion requirements, and improves the quality of life. In addition, identification of erythropoietin receptor (EpoR) expression on many types of non-erythroid and cancer cells has spurred an interest in the extra-haematological activities of Epo itself and other erythropoiesis-stimulating agents (ESAs). Epo and its derivatives have emerged as major tissue-protective cytokines in ischaemic and degenerative damage of cardiovascular, neurological and renal diseases, while their angiogenetic and immunomodulatory properties indicate that their therapeutic potential may extend well beyond erythropoiesis alone. Both preclinical and clinical data, however, have suggested that they may contribute to tumour progression and prejudice survival when administered to anaemic cancer patients, though the results are equivocal and the assumed mechanisms by which tumour growth could be promoted are not fully understood. While these findings offer new perspectives, they nonetheless demand caution in the employment of ESAs. Further, well-designed experimental and clinical studies are warranted.

Objective: Defective circulating dendritic cells (DCs) have been described in systemic lupus erythematosus (SLE) and correlated with high levels of interferon-alpha (IFNalpha). DCs are differentiated as being either myeloid or plasmacytoid, according to chemokine expression and the tendency to migrate toward inflamed tissue. We investigated the potential role of interleukin-18 (IL-18) in driving the glomerular migration of DCs in lupus nephritis (LN) and in affecting the ability of DCs to induce an imbalance in the Th1:Th2 ratio., Methods: DC subsets were characterized by flow cytometry and defined as either myeloid or plasmacytoid according to the expression of CD11c/blood dendritic cell antigen 1 (BDCA-1) and CD123/BDCA-2, respectively. The serum Th1:Th2 profile was studied by enzyme-linked immunosorbent assay. IL-18 receptor (IL-18R) and other chemokine receptors were analyzed by flow cytometry. Glomerular levels of IL-18/IL-18R and the presence of plasmacytoid DCs and myeloid DCs were investigated by immunohistochemical analysis., Results: The number of peripheral plasmacytoid DCs was decreased in patients with SLE compared with control subjects, and this defect in the number of DCs was correlated with LN. Patients with LN showed a prevalent Th1 response, with high production of IL-18, IL-12 and IFNgamma. Only plasmacytoid DCs expressed IL-18R. Patients with severe LN showed a high accumulation of IL-18 within glomeruli in association with the presence of plasmacytoid DCs, whereas myeloid DCs were almost absent., Conclusion: A deficient number of peripheral plasmacytoid DCs correlated with high levels of Th1 cytokines and was associated with LN. Both serum and glomerular IL-18 were increased in LN. It is suggested that the high level of expression of IL-18R by peripheral plasmacytoid DCs allows the DCs to relocate within glomeruli under IL-18 stimulation and triggers the resident T cells, thus promoting renal damage.

Hyperactive osteoclastogenesis is a hallmark of multiple myeloma, a B cell neoplasia homing to bone marrow and resulting in multiple osteolytic lesions and skeleton devastation. We provide evidence that myeloma cells can themselves act as osteoclasts in vitro. By extending standard cultures of U-266 and MCC-2 myeloma cell lines, we found that subsets of adherent cells also expressed the osteoclast phenotype, including multinuclear morphology, cytoplasmic tartrate-resistant acid phosphatase, the calcitonin receptor and a specific osteoclast antigen. These subsets resorbed bone substrates by producing osteoclast enzymes as well as the characteristic redistribution of F-actin in their cytoskeleton, thus forming the sealing zone that is adopted by adherent osteoclasts to generate the acidified environment essential for bone resorption. Neither the phenotype nor the functional properties of osteoclasts were detected in parental non-adherent cells. In adherent cultures osteoclastogenesis was associated with deregulated expression of both receptor activator of nuclear transcription factor (NF)-kappaB (RANK) and its ligand RANK-L, which triggers cell maturation in osteoclast precursors. Resorption of bone substrates was prevented by a neutralising anti-RANK-L antibody. Our data indicate that osteoclast-like transformation of both U-266 and MCC-2 cellular models of human myeloma is dependent on RANK-L stimulation.

Bone remodelling is severely affected in myeloma bone disease as a consequence of skeletal metastatization of malignant plasma cells. We investigated whether defective bone replacement is dependent on increased osteoblast apoptosis and/or on deregulated events within the bone microenvironment. Circulating tumour necrosis factor (TNF)-alpha, interferon-gamma, interleukin (IL)-1beta, and IL-6 levels were higher in myeloma patients with overt bone disease, whose osteoblasts constitutively overexpressed Fas, DR4/DR5 complex as receptors to TNF-related apoptosis inducing ligand, intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1). They were functionally exhausted and promptly underwent apoptosis in vitro, in contrast to the minor tendency to death detected in control osteoblasts from patients without bone involvement and normal donors. Osteoblasts dramatically enhanced their apoptosis in co-cultures with MCC-2 myeloma cells and upregulated both ICAM-1 and MCP-1 in a manner similar to control osteoblasts. Pretreating MCC-2 cells with soluble ICAM-1 led to a striking inhibition of their adhesion to osteoblasts, suggesting that the ICAM-1/lymphocyte function-associated antigen-1 system plays a role in the reciprocal membrane contact to trigger apoptogenic signals. Our data suggest that, in the myeloma bone microenvironment, both high cytokine levels and physical interaction of malignant plasma cells with osteoblasts drive the accelerated apoptosis in these cells leading to defective new bone formation.

Typical features of multiple myeloma (MM) are osteolytic lesions and severely affected bone regeneration. This study of 53 MM patients demonstrates an enhancement of osteoblast cytotoxicity by malignant myeloma cells via the upregulation of apoptogenic receptors, including Fas ligand (Fas-L) and tumour-necrosis-factor-related apoptosis inducing ligand (TRAIL). Both were significantly increased in the marrow myeloma cells of patients with extensive osteolytic lesions in a fashion similar to the highly malignant human myeloma cell line MCC-2. Osteoblasts from these subjects over-expressed Fas and death receptor (DR) 4/5 and underwent dramatic apoptosis when co-cultured with either MCC-2 or autologous myeloma cells. In osteoblast and myeloma cell co-cultures, monocyte chemoattractant protein 1 (MCP-1) mRNA was upregulated in osteoblasts from patients with severe bone disease in parallel with increased CC-chemokine receptor R2 (CCR2) expression, the ligand of MCP-1, in the myeloma cells. This chemokine was shown to activate malignant cell migration in vitro. An upregulation of ICAM-1 expression occurred in osteoblasts from patients with active skeleton disease. This upregulation appeared to be an effect of malignant plasma cell contact, as MCC-2 co-culture greatly enhanced ICAM-1 production by resting osteoblasts from patients without skeleton involvement. Our results suggest that osteoblasts in active myeloma are functionally exhausted and promptly undergo apoptosis in the presence of myeloma cells from patients with severe bone disease. It is suggested that this cytotoxic effect plays a pivotal role in the pathogenesis of defective bone repair.

We previously showed that serum beta-2-microglobulin (beta2m)-free human leucocyte antigen (HLA) class I heavy chain (FHC) levels were increased in MM and correlate with disease activity. The present investigation, carried out in 124 multiple myeloma patients, studied the expression of the three size variants of FHC, namely the 42 kDa intact heavy chain (A variant, AV), released through a shedding process, and the truncated FHC (tFHC) 39 kDa (BV) and 36-35 kDa (CV) released by means of membrane-type metalloprotease activity. The increase in FHC correlated with a high expression percentage of BV (r = 0.32, P = 0.0002) and tFHC (r = 0.42, P < 0.0001), which could help to discriminate multiple myeloma from monoclonal gammopathy of undetermined significance (tFHC mean ratio = 3.2; Mann-Whitney U-test, P < 0.0001). tFHC levels highly correlated with other disease activity markers, namely haemoglobin (r = -0.35; Spearman's rank, P = 0.0001), percentage of bone marrow plasma cells (r = 0.4, P < 0.0001) and beta2m levels (r = 0.36, P < 0.0001), while only the last barely correlated (r = 0.2, P = 0.03) with AV. Finally, the 0.4, 0.57 and 0.71 mg/l BV, tFHC and (to a lesser extent) FHC cut-off values divided patients into two groups with different survival curves (P = 0.0005, P = 0.0025 and P = 0.04 respectively). These data are in favour of a correlation between disease aggressiveness and cleavage of these variants by membrane-type metalloprotease enzymes.

Interleukin 6 (IL-6) is an important survival and growth factor for myeloma cells and exerts its effects by activating several transduction pathways, including the Ras cascade. As farnesylation of the activated Ras oncogene product by protein farnesyltransferase (FTase) is a critical step for Ras functional activity, FTase has emerged as a potential target for the development of new anti-cancer agents. Based on our previous demonstration that IL-6-producing myeloma cells are refractory to drug-induced apoptosis, we have analysed the effect of manumycin, a natural FTase inhibitor, on IL-6-producing myeloma cells resistant to Fas-, dexamethasone- and doxorubicin-induced apoptosis. Treatment of myeloma cells with manumycin prevented cell proliferation and induced apoptosis. Western blotting experiments demonstrated that this effect was related to inhibition of the post-translational Ras processing.Further analysis showed that manumycin-induced apoptosis involved caspase-3. Activation of caspase-3, in fact, was observed in 6 h-treated myeloma cells expressing Apo 2.7 antigen, the marker of early apoptosis, whereas their treatment with cell-permeable DEVD-fmk, that irreversibly inhibits caspase-3 activity, prevented their apoptosis. Over-expression of caspase-3 was also demonstrated by reverse transcription-polymerase chain reaction. Finally, over-expression of Bcl-2 and its homologue Bcl-xL was observed in manumycin-treated cells as well as in control myeloma cells, implying that the Bcl-2 family is not involved. FTase inhibitors may thus be proposed as a potential pharmacological weapon, as they block the Ras pathway and induce the apoptosis of drug-resistant IL-6-producing myeloma cells.

The erythrocyte water channel aquaporin 1 (AQP1) is expressed in multiple absorptive and secretory epithelia including the capillary endothelia. Immunoblot analysis showed that bone marrow biopsies of patients with active multiple myeloma (MM) display significantly higher levels of AQP1 than those from patients with non-active MM, whose values are higher, but to a lesser extent, than those of patients with monoclonal gammopathies of undetermined significance (MGUS). Values of MGUS overlapped those of patients with anaemia as a result of iron or vitamin B12 deficiencies (called 'benign anaemias'). Immunohistochemistry and computerized image analysis of AQP1 highlighted bone marrow microvessels whose area per microscopic field was significantly greater in patients with active MM, and always larger than and closely correlated with the microvessel area when assessed with factor VIII-related antigen/von Willebrand's factor (FVIII-VWF). The intensity of AQP1 expression by microvessels evaluated using image analysis was significantly greater in active than non-active MM and in the latter over MGUS or benign anaemias. It is suggested that, among plasma cell tumours, AQP1 expression is preferentially associated with microvessels of MM and that the highest degree of expression occurs in active MM in step with enhanced angiogenesis, in which AQP1 recognizes more immature neovessels than FVIII-VWF. It may, perhaps, favour angiogenesis in a positive loop and, hence, MM progression, and thus be applied for therapeutic vascular targeting.

Effects of epoetin alfa on transfusions, haemoglobin (Hb) and quality of life (QOL) were evaluated in a placebo-controlled study of 145 patients with multiple myeloma and anaemia (Hb < 11 g/dl). During the 12-week, double-blind phase, patients received 150 IU/kg epoetin alfa or a matching volume of placebo subcutaneously three times weekly; the dose (or volume) was doubled at week 4 if Hb response was inadequate. Patients completing this phase could enter the subsequent optional 12-week phase of open-label epoetin alfa treatment. During double-blind treatment, epoetin alfa significantly decreased the incidence of transfusion compared with placebo (28% vs. 47%, P = 0.017), regardless of patients' transfusion history, and increased mean Hb (1.8 g/dl vs. 0.0 g/dl, P < 0.001). Univariate analysis showed significant (P

Serum levels of beta2-microglobulin (beta2m)-free HLA class I heavy chain (FHC) in 94 patients with multiple myeloma (MM) were higher than in 29 patients with monoclonal gammopathy of undetermined significance (MGUS) (P = 0.023) and in 97 sex- and age-matched healthy controls (P < 0.0001). Spearman correlation analysis indicated that in MM, FHC correlated with beta2m (r = 0.31, P = 0. 003) and the percentage of bone marrow plasma cells (BMPC%) (r = 0. 36, P = 0.002), whereas beta2m, in addition to BMPC% (r = 0.43, P = 0.0003), also correlated with creatinine levels (r = 0.63, P < 0.0001), haemoglobin levels (r = -0.35, P = 0.0007) and patient age (r = 0.34, P < 0.0011). Furthermore, MM patients with poor prognosis (beta2m >/= 6 mg/l) displayed higher FHC levels than those with a better prognosis (beta2m < 6mg/l) (P < 0.021). At variance from beta2m, these levels were not influenced by renal failure, as indicated by the lack of Spearman correlation of FHC with creatinine concentration and of statistical significance between the median FHC concentration of MM patients with creatinine < 176.6 micromol/l and those with creatinine >/= 176.6 micromol/l (P = 0.3). Stratification of patients according to disease activity and stage showed that FHC levels were only statistically different (P = 0.04) for disease activity, whereas beta2m and C-reactive protein were not. Taken together, our data indicate that serum FHC may be a useful disease marker in MM.

Objective: To investigate whether immunologic abnormalities in patients with Behçet's disease (BD) are related to abnormalities of the Th1/Th2 ratio., Methods: Th1/Th2 cytokine production by peripheral blood lymphocytes (PBL) from 31 patients with BD, 11 patients with inflammatory arthritis, and 10 healthy blood donors was evaluated by intracellular immunofluorescence staining. Serum interleukin-12 (IL-12) levels were measured using an enzyme amplified-sensitivity immunoassay. The effect of recombinant IL-12 (rIL-12) on spontaneous and Fas-mediated apoptosis of phytohemagglutinin (PHA)-stimulated PBL was evaluated by flow cytometry using propidium iodide (PI) staining and a bromodeoxyuridine (BrdU)/PI procedure., Results: Intracellular immunofluorescence staining of IL-2, IL-4, and interferon-gamma (IFNgamma) in CD3+ lymphocytes from BD patients demonstrated a strong polarization of the immune response toward the Th1 pathway that correlated with the progression of BD. Peripheral Th1 cells were significantly increased in patients with active disease (n = 14) as compared with those in patients in complete remission (n = 17), patients with inflammatory arthritis, and normal donors. In addition, serum IL-12 levels were correlated with peripheral Th1 lymphocytes and disease progression. Apoptotic analysis revealed that PHA-activated PBL from patients with active disease were highly sensitive to spontaneous and Fas-mediated activation-induced cell death. However, addition of rIL-12 to complete medium prevented this spontaneous and Fas-induced apoptosis and enhanced the proliferation of Th1 lymphocytes., Conclusion: Taken together, these results indicate that a strong Th1 immune response occurs in active BD and suggest that IL-12 plays a substantial part in the pathogenesis of BD. By preventing spontaneous and Fas-induced cell death, in fact, it results in an abnormal growth of autoreactive Th1 lymphocytes that could contribute to the prolonged inflammatory autoimmune condition of BD.

The aim of this study was to evaluate the role of CD8+/CD57+ lymphocytes in the immune dysregulation of multiple myeloma (MM). Cytofluorimetry of peripheral blood lymphocytes (PBL) purified from 39 MM patients showed an inverse relationship between the percentage of CD8+/CD57+ cells and CD4/CD8 ratio. Analysis of their activation antigens revealed that they were prevalently HLA-DR+ and Fas+. Removal of CD8+/CD57+ cells from MM PBL significantly improved cell proliferation and pokeweed mitogen (PWM)-induced polyclonal Ig production in vitro, whereas the addition of supernatants from patients' CD8+/CD57+ cell cultures to normal PBL suppressed both the PWM-driven Ig synthesis and the proliferative rate of stimulated PBL, supporting the contention that CD8+/CD57+ cells release in vitro an inhibitory factor that is directly involved in T-cell regulatory function. However, since the proliferative recovery of PWM- and phytohaemagglutinin (PHA)-stimulated MM PBL in the absence of CD8+/CD57+ lymphocytes was only partial, a dysregulated activation-induced apoptosis was anticipated. In fact, patients' PBL displayed an increased susceptibility to apoptosis and this was significantly enhanced after PWM and, even more, after PHA stimulation. Analysis of CD57 antigen expression on apoptotic or viable cells demonstrated a substantial defect of apoptosis in the CD8+/CD57+ population. Our results indicate that both the immunosuppressive effect of CD8+/CD57+ cells and the enhanced susceptibility to apoptosis of PBL could be involved in the pathogenesis of the immunodeficiency observed in this disease.

We studied the molecular alterations of IL-6R, lck and c-myc genes in the tumour cells of 50 patients with multiple myeloma (MM) and 20 patients with monoclonal gammopathies of undetermined significance (MGUS). Southern blot analysis revealed a 5.3 kb IL-6R amplified band by digestion with EcoRI and HindIII in three MGUS patients, but no IL-6R gene alteration was found in MM patients. BamHI digestion revealed a 6.2 kb rearranged band of the lck gene in two MGUS patients with IL-6R amplification. In one MGUS patient we detected a rearrangement upstream of the lck coding region. Myc rearrangement was observed in three MM and two MGUS patients who showed coexistent lck rearrangement and IL-6R amplification. These molecular alterations were detected in the MGUS patients with an IgA monoclonal component, who showed a rapid progression into aggressive MM. Myc rearrangement seems to be associated with a small group of a clinically aggressive MM at diagnosis, secreting IgA or k light chains. Multiple rearrangements and/or molecular alterations are likely to occur at the time of MGUS-IgA transformation into aggressive MM and myc rearrangement may promote a positive pressure for transformation and progression. MM tumourigenesis remains a heterogenous multistep process involving the deregulation of many genes and gene products.

Bone marrow plasma cells and stromal cells in multiple myeloma (MM) have been shown to be capable of releasing cytokines with angiogenic properties. Plasma cells can also express adhesion molecules controlling their adhesive interactions with endothelial cells. In the present study, we have evaluated by immunohistochemistry the extent of angiogenesis in the bone marrow of: a) 51 patients with active and non-active MM; b) 25 patients with monoclonal gammopathy of undetermined significance (MGUS). Plasma cells were investigated by flow cytometry for the expression of the adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. The results showed that, while angiogenesis was very low or absent in patients with MGUS and non-active MM, it increased markedly in those with active MM. The highest detectability of plasma cell adhesion molecules, except LAM-1, was also found in these patients. The functional significance of these findings is unknown. Their consistent occurrence in the bone marrow of active myeloma patients, however, strongly suggests that more frequent adhesive interactions between plasma cells and their microvasculature underlie tumor dissemination.

We investigated the prognostic significance of interleukin-10 (IL-10) and soluble interleukin-2 receptor (sIL-2r) levels in the pretreatment serum of 105 individuals with newly-diagnosed aggressive non-Hodgkin's lymphoma (NHL). Commercially available enzyme-linked immunoassay kits were used for cytokine and receptor measurements. Detectable levels of IL-10 were found in 42 (40%) patients at diagnosis, with no correlation with clinico-haematological parameters, but in no control samples (P < 0.001). Pretreatment concentrations of sIL-2r were markedly increased in individuals with NHL when compared to controls (2614 +/- 893 U/ml v 219 +/- 65 U/ml, P < 0.001), patients with stage III/IV presenting higher values than those with stage II disease (3885 +/- 1196 U/ml v 1732 +/- 646 U/ml, P < 0.001). No single parameter was associated with the achievement of complete remission, but the combination of elevated IL-10 and of sIL-2r greater than 3000 U/ml selected a subset of patients with a high probability of failing induction therapy (P < 0.001). Life-table analysis also indicated that patients with these characteristics have a significantly shorter event-free survival. In a multivariate analysis the combination of IL-10 with sIL-2r was found to have greater predictive strength than the combination of IL-10 with beta 2-microglobulin. We conclude that IL-10 and sIL-2r measurements can be expected to improve existing methods of risk assignment in aggressive NHL.

Tumour growth is angiogenesis-dependent. We found a high correlation between the extent of bone marrow angiogenesis, evaluated as microvessel area, and the proliferating (S-phase) fraction of marrow plasma cells, evaluated as labelling index (LI), in patients with multiple myeloma (MM) and in those with monoclonal gammopathies of undetermined significance (MGUS). Angiogenesis itself was significantly associated with active as opposed to non-active MM and MGUS. The highest microvessel area accompanied rapidly progressive MM with the highest LI. When a cut-off value of 2% or greater of the microvessel area was used, most patients with active MM were classified correctly. The risk of active disease in patients with MM increased in parallel with the microvessel area. A causal relationship between plasma cell growth, activity phase in MM and marrow angiogenesis is suggested. Since angiogenesis proceeds in step with the enlargement of plasma cell tumours and the activity phase in MM, its measurement could be a useful prognostic marker in patients with plasma cell proliferative disorders.