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Objective.Neutrophil extracellular traps (NET) expose modified antigens for autoantibodies in vasculitis. Little is known about levels and removal pathways of NET in systemic lupus erythematosus (SLE), especially in lupus nephritis (LN). We determined circulating levels and defined NET removal in large subsets of patients with incident SLE (iSLE), some of whom had new-onset nephritis.Methods.Serum levels of NET (ELISA), DNase1/DNase1L3 (ELISA), and DNase activity (functional assay) were determined in 216 patients with iSLE [103 had incident LN (iLN)], in 50 patients with other primary glomerulonephritis, and in healthy controls. Ex vivoNET production by neutrophils purified from a random selection of patients was quantified as elastase/DNA release and by immunofluorescence techniques.Results.Serum NET levels were very high in iSLE/iLN compared to all groups of controls and correlated with anti-dsDNA, C3–C4, and proteinuria; iLN had the highest levels. DNase activity was decreased in iLN compared to SLE (20% had one-half DNase activity) despite similar serum levels of DNase1/DNase1L3. In these cases, pretreatment of serum with protein A restored DNase efficiency; 1 patient was homozygous for a c.289_290delAC variant of DNASE1L3. Ex vivoNET production by neutrophils purified from LN, SLE, and normal controls was similar in all cases.Conclusion.Patients with iLN have increased circulating NET and reduced DNase activity, the latter being explained by the presence of inhibitory substances in circulation and/or by rare DNase1L3mutations. Accumulation of NET derives from a multifactorial mechanism, and is associated and may contribute to disease severity in SLE, in particular to renal lesions. (Clinical trial registration: The Zeus study was registered at ClinicalTrials.gov, study number NCT02403115).

ABSTRACTIntroduction: TNF-α inhibitors can be administered either as monotherapy or in combination with other anti-inflammatory drugs or DMARDs in the treatment of chronic immune-mediated diseases.Areas covered: Patients receiving TNF-α inhibitors are at high risk of infections. An update is made on the risk of infection in patients receiving TNF-α inhibitors and the strategies for mitigating against the development of these serious adverse events.Expert opinion: Infliximab than etanercept appears to be responsible for the increased risk of infections. Re-activation of latent tuberculosis infection and the overall risk of opportunistic infections should be considered before beginning a course of TNF-α inhibitors. A careful medical history, Mantoux test/quantiferon-TB Gold In-tube Test and chest-X-ray should always be performed before starting TNF-α inhibitors. Particular attention should be paid to risk factors for Pneumocystis jirovecii infection. Hepatitis B and C virological follow-up should be considered during TNF-α inhibitors treatment. Finally, appropriate vaccinations for influenza, S. pneumoniae, and HBV should be administered to decrease the risk of infection, and patients who are at high risk of herpes zoster reactivation would benefit from a second vaccination in adulthood.

ABSTRACTIntroduction: TNF-α inhibitors have demonstrated efficacy both as monotherapy and in combination with disease-modifying anti-rheumatic drugs (DMARDs) in the treatment of chronic inflammatory immune-mediated diseases. Etanercept is a decoy receptor” for TNF-α and it is composed of two p75 TNF-α receptors fused to human IgG1.Areas covered: We discuss the potential role of pharmacogenetics in predicting the response to etanercept in patients with Ps and PsA.Expert opinion: Pharmacogenetics represents the new frontier for the discovery of potential genetic markers of biological response to TNF-α inhibitors. Clinical studies showed that TNF-α −308 G/G, +489 GG and the +489 GA, TNF-α −857C (rs1799724), TNFRSF1B 676T (rs1061622), TNFAIP3 G SNP (rs610604), FcγRIIIA-V158F, HLA-C*06, IL-17 A (rs2275913 and rs10484879), IL-17F (rs763780) and IL17RA (rs4819554) SNPs favor the response to etanercept. However, most of these studies are often small and not sufficiently powered to detect an effect and markers tend to be more prognostic than predictive of therapeutic response. Furthermore, studies often examines only the effects of a single SNP, while it would be more useful to analyze more haplotypes in contemporary in the same patients. Appropriately designed clinical trials are needed before a pharmacogenetic approach may be applicable in daily clinical therapeutic practice.

Objective To investigate the effects of long-term treatment with bosentan on pulmonary arterial hypertension (PAH) in patients with systemic sclerosis.Methods Patients with systemic sclerosis were followed between 2003 and 2014; those who developed digital ulcers were treated with standard regimens of bosentan. Patients were assessed at baseline and every 12 months using transthoracic Doppler echocardiography, 6-min walking distance test, Borg dyspnoea index and monitoring of plasma levels of 76-amino-acid N-terminal probrain natriuretic peptide. Patients who developed PAH underwent right heart catheterization to confirm the diagnosis.Results Sixty-nine patients with systemic sclerosis were enrolled in the study. Of these, 25 developed digital ulcers and received treatment with bosentan; the remaining 44 comprised the control group. None of the patients treated with bosentan developed PAH during the follow-up period. Furthermore, in these patients the mean ± SD systolic pulmonary arterial pressure significantly decreased from 33.64 ± 2.91 mmHg at baseline to 26.20 ± 1.78 mmHg at the end of the follow-up period. In contrast, in the control group, seven patients developed PAH during the follow-up period, with the mean ± SD systolic pulmonary arterial pressure significantly increasing from 33.57 ± 2.75 mmHg at baseline to 39.41 ± 4.11 mmHg at the end of the follow-up period.Conclusion Long-term treatment with bosentan reduces the risk of developing PAH in patients with systemic sclerosis.

Systemic Lupus Erythematosus (SLE) is characterized by abnormal autoantibody production and clearance. Infections are among the most important causes of morbidity and mortality in SLE patients; they have an increased frequency of severe bacterial and viral infections possibly due to inherited genetic and immunologic defects and to immunosuppressive therapies. In addition, infectious agents can switch on lupus disease expression and activity. Among the strategies to reduce the risk of infection, vaccination can be considered the most reliable option. Most vaccines are effective and safe in SLE patients, although in certain cases immunogenicity may be sub-optimal and vaccination can trigger a flare. Although these issues are currently unresolved, the risk benefit balance is in favor for vaccination to reduce the risk of infection in SLE patients. In the present review we discuss the preventive strategies currently recommended to reduce bacterial and viral infections in SLE.

Introduction:TNF-α is a pro-inflammatory cytokine known to a have a key role in the pathogenesis of chronic immune-mediated diseases. TNF-α inhibitors can be administered either as monotherapy or in combination with other anti-inflammatory or disease-modifying anti-rheumatic drugs (DMARDs) to treat chronic immune-mediated diseases.Areas covered:Patients receiving TNF-α inhibitors are at high risk of infections. Based on our experience, in this paper, we discuss the risk of infections associated with the administration of TNF-α inhibitors and the strategies for mitigating against the development of these serious adverse events.Expert opinion:Infliximab more so than etanercept appears to be responsible for the increased risk of infections. Re-activation of latent tuberculosis (LTB) infection and the overall risk of opportunistic infections should be considered before beginning TNF-α inhibitor therapy. A careful medical history, Mantoux test and chest-x-ray should always be performed before prescribing TNF-α inhibitors. Particular attention should be paid to risk factors for Pneumocystis jirovecii infection. Hepatitis B and C virological follow-up should be considered during TNF-α inhibitor treatment. Finally, patients who are at high risk of herpes zoster (HZ) reactivation would benefit from a second vaccination in adulthood when receiving TNF-α inhibitors.

Introduction:During the last decade, many new biological immune modulators have entered the market as new therapeutic principles. Biologics, including TNF-α inhibitors, are the new frontier in the treatment of immune-mediated or inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Crohn’s disease, ankylosing spondylitis, systemic sclerosis, disseminated granuloma annulare, psoriasis and/or psoriatic arthritis. TNF-α inhibitors have demonstrated efficacy and are well tolerated in large, randomized, controlled clinical trials. However, a substantial proportion of patients do not respond to these agents and potential adverse drug reactions may be associated with its use.Areas covered:Pharmacogenetics has the potential of increasing drug efficiency by identifying genetic factors responsible for lack of response or toxicities to TNF-α inhibitors. In this review, we analyze the influence of several polymorphisms upon the efficacy and safety of TNF-α inhibitors.Expert opinion:Several polymorphisms have been proven to influence the response to etanercept. Among them, single nucleotide polymorphisms (SNPs) −308 G/G, −857 C/T, 489 GG and GA, HLA-DRB1-encoding SE (allele 0404 and allele 0101) favor the response to etanercept, whereas SNP −308 A/A and TNFR1A AA decrease the response. Large clinical studies are needed to confirm the relevance of these associations in order to tailor treatment and to decrease unnecessary toxicity.

Introduction:TNF-α inhibitors have demonstrated efficacy in large, randomized controlled clinical trials either as monotherapy or in combination with other anti-inflammatory or disease-modifying antirheumatic drugs in the treatment of chronic inflammatory immune-mediated diseases. Etanerceptis a fusion protein that acts as a ‘decoy receptor’ for TNF-α.Areas covered:This paper evaluates the efficacy and safety of etanercept in patients with chronic inflammatory immune-mediated diseases.Expert opinion:Etanercept was first approved for the treatment of rheumatoid arthritis (RA) and subsequently of chronic plaque psoriasis, psoriatic arthritis, ankylosing spondylitis and juvenile RA. Etanercept as other TNF-α inhibitors, particularly infliximab, may be administered off-label to treat other chronic inflammatory immune-mediated diseases such as systemic sclerosis, Behcet disease, systemic lupus erythematosus, polymyositis, dermatomyositis and mixed connective tissue disease. Early etanercept treatment prevents joint damage and helps to avoid long-term disability in arthritis. Etanercept administered at a dose of 50 mg once weekly is effective in inducing an earlier remission of RA, and etanercept 50 mg twice weekly may favor a more rapid improvement of psoriasis and psoriatic arthritis. Etanercept and adalimumab may exert beneficial effects on lipid profile and improve endothelial dysfunction. Appropriate screening tests for latent tuberculosis, hepatitis B virus and hepatitis C virus should be performed before starting etanercept. TNF-α inhibitors including etanercept are contraindicated in patients with demyelinating diseases.

Introduction:Systemic sclerosis (SSc) is a rare connective tissue disease characterized by chronic inflammation and fibrosis of the skin, vascular abnormalities and variable involvement of organs. Patients with limited SSc typically develop pulmonary arterial hypertension (PAH). TNF-α, VEGF, platelet-derived growth factor and endothelin-1 play a key role in the development of PAH.Areas covered:This paper addresses the efficacy and safety of current drugs used for the treatment of PAH.Expert opinion:Bosentan, ambrisentan, sildenafil, tadalafil, iloprost, epoprostenol and treprostinil were associated with hemodynamic improvements in PAH patients. Ambrisentan has a better safety profile compared with bosentan, regarding the risk of increase in hepatic transaminases. Flushing, dyspepsia and diarrhea were the most frequent adverse events in patients treated with sildenafil, while headache, myalgia and flushing were the adverse events in those receiving tadalafil. Inhaled iloprost is also effective, but it requires multiple daily nebulizations up to 15 min each and may induce cough, flushing, jaw pain and headache. Epoprostenol is considered the most effective approved therapy for severe PAH in WHO functional class III and class IV. TNF-α inhibitors reduce the systemic inflammation in patients with chronic immune-mediated diseases and improve the endothelial function, decreasing the risk of PAH progression.

Rheumatoid arthritis (RA) is a chronic inflammatory immune-mediated systemic disease which primarily affects the joints. Leflunomide (LFN) is a disease modifying anti-rheumatic drugs (DMARDs) which acts by inhibiting the synthesis of pyrimidines. Several trials have demonstrated the efficacy and safety of LFN alone or in combination with biological agents such as tumor necrosis factor-α (TNF-α) inhibitors in the treatment of RA patients. TNF-α is a pro-inflammatory cytokine known to a have a key role in the pathogenesis of RA. TNF-α inhibitors have demonstrated efficacy as monotherapy or in combination with other anti-inflammatory or DMARDs in the treatment of RA. Five TNF-α inhibitors are available for clinical use and include infliximab, adalimumab, golimumab, certolizumab pegol and etanercept. In this editorial, we briefly discuss the efficacy and safety of LFN and TNF-α inhibitors in the treatment of RA, and the potential beneficial effect of both LFN and TNF-α inhibitors in improving the endothelial dysfunction and in reducing the risk of acute cardiovascular and/or cerebrovascular events.

Introduction:During the last decade, many new biological immune modulators entered the market as new therapeutic principles. TNF-α is a pro-inflammatory cytokine known to a have a key role in the pathogenic mechanisms of various immune-mediated or inflammatory diseases. TNF-α blockers have demonstrated efficacy in large, randomized controlled clinical trials either as monotherapy or in combination with other anti-inflammatory or disease-modifying anti-rheumatic drugs.Areas covered:Although generally well tolerated and safe, potential adverse events may be associated with TNF-α inhibitor treatment. The authors will briefly review the potential adverse drug reactions and the immunological mechanisms of injection site reactions (ISRs) in patients treated with etanercept and adalimumab. Expert opinion:Patients treated with TNF-α inhibitors can develop ISR around the sites of injections. ‘Type IV delayed type reaction' or ‘recall ISRs'. Eosinophilic cellulitis or ‘Wells syndrome', ‘Type III' and ‘Type I' reactions are reported. Long-term studies are necessary to determine the durability of response and the real risk of ISRs with golimumab and certolizumab pegol. Further studies are also necessary to evaluate the immunogenicity of these drugs.

The ongoing progresses in the knowledge of the pathogenic mechanisms of various immune-mediated and inflammatory diseases as well as the availability of innovative biotechnological approaches have led to the development of new drugs that add to conventional treatments. Among these, tumor necrosis factor (TNF)-α inhibitors, that is, infliximab, adalimumab, etanercept, golimumab and certolizumab pegol, are now available for clinical use. This editorial discusses the recent indications of TNF-α inhibitors, the pretreatment considerations, the reported adverse events and, finally, the recommendations for its use in pregnancy.

OBJECTIVE: Headache in systemic lupus erythematosus (SLE) is controversial, as is evidence of brain impairment in patients with SLE and headache. Perfusion single photon emission computed tomography (SPECT) was performed to investigate brain impairment in SLE patients with migraine-like headache either from the period of diagnosis or later in the course of disease. METHODS: Eighteen patients with SLE (mean age 40.8 ± 13.6 yrs) matching these characteristics underwent brain SPECT with 99mTc-HMPAO in the interictal period. Electroencephalography (EEG) and magnetic resonance imaging (MRI) were performed in 12 and 10 patients, respectively. SPECT was analyzed through visual and asymmetry combined analysis as well as by voxel-based statistical analysis compared to a control group of matched healthy subjects (statistical threshold: p = 0.01). RESULTS: Focal hypoperfusion was evidenced in 15 (83%) patients, often matching the main side of pain location, whereas EEG and MRI each gave a positive result in 50% of cases. Using voxel-based analysis, significant hypoperfusion was found in 8 (44%) patients, either lateralized to one side or localized to the anterior cingulate cortex, independent of pain location. CONCLUSION: Brain perfusion SPECT is a sensitive tool for identifying brain impairment in SLE-related migraine, although the mechanisms of brain damage remain to be elucidated. Besides confirming focal hypoperfusion in some patients, in 4 patients statistical analysis revealed interictal hypofunction of the anterior cingulate cortex, a key structure for cortical elaboration of pain in the midline network.

Subcutaneous specific immunotherapy has been demonstrated to be capable of inducing T-cell regulatory response. Interleukin 10 (IL-10) plays a crucial role in inducing allergen-specific tolerance; however, no previous studies have examined IL-10 production after sublingual immunotherapy (SLIT).

Studies comparing transfusion and nontransfusion patients suggest an increased risk of postoperative infections in transfusion groups. Supernatants of blood components have been shown to affect the function of T lymphocytes and natural killer cells. Here, we found that supernatants from stored red blood cells (RBCs) inhibit human neutrophil migration in response to formyl peptides and stimulate neutrophil locomotion. These effects can be observed with high dilutions of RBC supernatants, such as 1:5 × 106 (vol/vol), able to trigger locomotion as well as desensitization of the cells to alternative chemoattractants. The phenomenon might be mediated by chemoattractants present in the supernatants. As RBC supernatants failed to mobilize intracellular free calcium, the chemoattractants should belong to the group of pure chemoattractants, that is, soluble Fas ligand (sFasL) and transforming growth factor–β1 (TGF-β1), known to act without increasing calcium levels. Recombinant TGF-β1, but not sFasL, was found to reproduce the ability of RBC supernatants to both inhibit neutrophil response to formyl peptides and stimulate neutrophil locomotion. Moreover, TGF-β1–immunodepleted supernatants did not display neutrophil-directed activities. Finally, RBC supernatants from RBCs stored after depletion of leukocytes were incapable of affecting neutrophil function. With neutrophils acting as a first-line antimicrobial defense, the ability, shown here, of high dilutions of RBC supernatants to inhibit neutrophil chemotaxis through TGF-β1 may be a relevant determinant of infections in the postoperative period for transfusion patients. Consistently, the neutrophil chemotactic response to formyl peptide was inhibited by the plasma obtained from 5 transfusion patients.

Studies comparing transfusion and nontransfusion patients suggest an increased risk of postoperative infections in transfusion groups. Supernatants of blood components have been shown to affect the function of T lymphocytes and natural killer cells. Here, we found that supernatants from stored red blood cells (RBCs) inhibit human neutrophil migration in response to formyl peptides and stimulate neutrophil locomotion. These effects can be observed with high dilutions of RBC supernatants, such as 1:5 × 106(vol/vol), able to trigger locomotion as well as desensitization of the cells to alternative chemoattractants. The phenomenon might be mediated by chemoattractants present in the supernatants. As RBC supernatants failed to mobilize intracellular free calcium, the chemoattractants should belong to the group of pure chemoattractants, that is, soluble Fas ligand (sFasL) and transforming growth factor–β1 (TGF-β1), known to act without increasing calcium levels. Recombinant TGF-β1, but not sFasL, was found to reproduce the ability of RBC supernatants to both inhibit neutrophil response to formyl peptides and stimulate neutrophil locomotion. Moreover, TGF-β1–immunodepleted supernatants did not display neutrophil-directed activities. Finally, RBC supernatants from RBCs stored after depletion of leukocytes were incapable of affecting neutrophil function. With neutrophils acting as a first-line antimicrobial defense, the ability, shown here, of high dilutions of RBC supernatants to inhibit neutrophil chemotaxis through TGF-β1 may be a relevant determinant of infections in the postoperative period for transfusion patients. Consistently, the neutrophil chemotactic response to formyl peptide was inhibited by the plasma obtained from 5 transfusion patients.

There is convincing evidence that soluble HLA-A,-B,-C (sHLA-A,-B,-C) and soluble HLA-G (sHLA-G) antigens can induce apoptosis in CD8+ activated T 3;cells although there is scanty and conflicting information about the mechanism(s) by which sHLA-A,-B,-C antigens and sHLA-G antigens induce apoptosis. In this study we have compared the apoptosis-inducing ability of sHLA-A,-B,-C antigens with that of sHLA-G1 antigens in CD8+ T 3;lymphocytes and CD8+ NK cells. Furthermore we have compared the inhibitory effect of sHLA-A,-B,-C antigens and of sHLA-G1 antigens on theactivity of EBV-specific CD8+ cytotoxic T lymphocytes (CTL). sHLA molecules were purified from serum and from the supernatant of HLA class 3;I-negative cells transfected with one gene encoding either classical or non-classical HLA class 3;I antigens. Both classical and non-classical sHLA class 3;I molecules trigger apoptosis in CD8+ T 3;lymphocytes and in CD8+ NK cells, which lack the T 3;cell receptor, and their apoptotic potency is comparable. The binding of sHLA-A,-B,-C and sHLA-G1 molecules to CD8 leads to Fas ligand (FasL) up-regulation, soluble FasL (sFasL) secretion and CD8+ cell apoptosis by Fas/sFasL interaction. Moreover, classical and non-classical sHLA class 3;I molecules inhibit the cytotoxic activity of EBV-specific CD8+ CTL. As the amount ofsHLA-G molecules detectable in normal serum is significantly lower than that of sHLA-A,-B,-C molecules, the immunomodulatory effects of sHLA class 3;I molecules purified from serum are likely to be mainly attributable to classical HLA class 3;I antigens. As far as the potential in vivo relevance of these findings is concerned, we suggest that classical sHLA class 3;I molecules may play a major immunoregulatory role in clinical situations characterized by activation of the immune system and elevated sHLA-A,-B,-C serum levels. In contrast, non-classical HLA class 3;I molecules may exert immunomodulatory effects in particular conditions characterized by elevated sHLA-G levels such as pregnancy and some neoplastic diseases.

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I–Cw4 or –Cw3 from untransfected or –Cw4 or –Cw3 alleles transfected HLA-A−, B−, C−, G−, E+, F+721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR– or anti-KIR–specific monoclonal antibodies (mAbs). After sHLA-I–activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I–mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor–mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H+–adenosine triphosphatase (H+-ATPase) of granules, inhibited activating receptor–induced NK cell cytolysis, suggesting that activating receptor–mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-γ (IFN-γ) was detectable in culture supernatant of activating receptor+NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor–mediated IFN-γ production. This suggests that activating receptor–induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-γ, an antiviral cytokine able to amplify immune responses.

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I–Cw4 or –Cw3 from untransfected or –Cw4 or –Cw3 alleles transfected HLA-A−, B−, C−, G−, E+, F+ 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR– or anti-KIR–specific monoclonal antibodies (mAbs). After sHLA-I–activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I–mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor–mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H+–adenosine triphosphatase (H+-ATPase) of granules, inhibited activating receptor–induced NK cell cytolysis, suggesting that activating receptor–mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-γ (IFN-γ) was detectable in culture supernatant of activating receptor+ NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor–mediated IFN-γ production. This suggests that activating receptor–induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-γ, an antiviral cytokine able to amplify immune responses.

Immunological tolerance can be achieved by several mechanisms including suppressor cells, soluble factors, and neurohormonal mediators. On the cellular level, we isolated a population of CD8CD28? T cells capable of inhibiting anti-CD3 mAb-induced proliferation of autologous peripheral blood mononuclear cells in an HLA-I nonrestricted manner via production of IFN-? and IL-6. Interestingly, CD8CD28? T cells from systemic lupus erythematosus patients with active disease do not display this inhibitory activity and show a marked imbalance between inhibitory (IL-6) and stimulatory (IL-12) cytokines. For soluble factors, we studied soluble HLA molecules (sHLAs) and double-stranded DNA (ds-DNA). Soluble HLA-I (sHLA-I) molecules induce soluble Fas ligand (sFasL) secretion and trigger apoptosis in phytohemagglutin (PHA)-activated Fas T cells. Double-stranded DNA binds to HLA-II molecules and inhibits HLA-II-mediated antigen presentation. On the neurohormonal side, we focused our attention on the immunological activity of corticosteroids (CTSs). CTSs inhibit recirculation of CD4 T cells, suppress the proliferation and immunological function of activated T cells, and induce apoptosis of activated lymphocytes. Taken together, these data suggest the presence of a complex network of immunoregulatory mechanisms in which CTSs play a strong role supporting their recognized efficacy in the treatment of inflammatory and immunological diseases.

Herein, we show that CD8dull, CD8intermediate, and CD8bright natural killer (NK) cell clones can be identified. Triggering of CD8 with its natural ligand(s), represented by soluble HLA class I (sHLA-I), isolated either from serum of healthy donors or from HLA-I− 721.221 lymphoblastoid cell line transfected with HLA-A2, -Cw4, and -Bw46 alleles, or HLA-G1 leads to NK cell apoptosis. The magnitude of this effect directly correlated with the level of CD8 expression. sHLA-I–induced apoptosis depends on the interaction with CD8, as it was inhibited by masking this molecule with specific monoclonal antibodies (mAbs). Moreover, sHLA-I or CD8 cross-linking with specific mAbs elicited intracellular calcium increases, Fas ligand (FasL) messenger RNA transcription, and FasL secretion, which were needed for delivering the death signal. Indeed, this apoptosis was inhibited by preincubation of NK cell clones with Fas or FasL antagonist mAbs, indicating that the Fas/FasL pathway is involved. Furthermore, members of the inhibitory receptor superfamily, such as CD94/NKG2 complex or killer inhibitory receptors, were shown to exert an inhibitory effect on sHLA-I–mediated apoptosis and secretion of FasL. These findings suggest that interaction between sHLA-I and CD8 evokes an apoptotic signal that is down-regulated by inhibitory receptor superfamily that function as survival receptors in NK cells.

Herein, we show that CD8dull, CD8intermediate, and CD8brightnatural killer (NK) cell clones can be identified. Triggering of CD8 with its natural ligand(s), represented by soluble HLA class I (sHLA-I), isolated either from serum of healthy donors or from HLA-I−721.221 lymphoblastoid cell line transfected with HLA-A2, -Cw4, and -Bw46 alleles, or HLA-G1 leads to NK cell apoptosis. The magnitude of this effect directly correlated with the level of CD8 expression. sHLA-I–induced apoptosis depends on the interaction with CD8, as it was inhibited by masking this molecule with specific monoclonal antibodies (mAbs). Moreover, sHLA-I or CD8 cross-linking with specific mAbs elicited intracellular calcium increases, Fas ligand (FasL) messenger RNA transcription, and FasL secretion, which were needed for delivering the death signal. Indeed, this apoptosis was inhibited by preincubation of NK cell clones with Fas or FasL antagonist mAbs, indicating that the Fas/FasL pathway is involved. Furthermore, members of the inhibitory receptor superfamily, such as CD94/NKG2 complex or killer inhibitory receptors, were shown to exert an inhibitory effect on sHLA-I–mediated apoptosis and secretion of FasL. These findings suggest that interaction between sHLA-I and CD8 evokes an apoptotic signal that is down-regulated by inhibitory receptor superfamily that function as survival receptors in NK cells.

The serum levels of soluble β2-μ-associated and β2-μ-free HLA class I heavy chains were determined in 28 interferon-α nonresponder chronic hepatitis C patients retreated with interferon-α plus ribavirin and in 70 healthy subjects. The baseline levels of β2-μ-associated and β2-μ-free HLA class I heavy chains were significantly higher in patients than in healthy controls(P = 0.001).The levels of β2-μ-associated HLA class I heavy chains significantly increased in responder patients with respect to nonresponders at the third month of treatment(P = 0.03).At the sixth month of treatment and after 6 months of follow up the levels of β2-μ-associated HLA class I heavy chains decreased in responder patients and increased in nonresponders. The levels of β2-μ-free HLA class I heavy chains showed only minor changes during and after treatment. We suggest that the determination of hepatitis C virus RNA levels combined with soluble β2-μ-associated HLA class I heavy chains, as a marker of immune activation, could identify interferon-α non responder chronic hepatitis C patients most likely to respond to a retreatment with interferon-α plus ribavirin.

Eight patients affected by non-small-cell lung cancer were treated with intralesional and systemic recombinant IL-2(rIL-2) injection with the aim of activating both tumour-infiltrating lymphocytes and circulating cytotoxic or killer cells. The schedule of treatment was as follows: a daily fine-needle transparietal intralesional rIL-2 injection (1×105 Cetus units) from day 1 to day 5 and systemic rIL-2 infusion (1×105 Cetus units kg-1 day-1) from day 6 to day 10. One to four cycles of treatment were received by each patient. Clinical and immunological evaluations were performed (a) before treatment, (b) following the intralesional rIL-2 administration, (c) 1 h after the beginning of rIL-2 infusion and (d) at the end of the systemic rIL-2 infusion. No complete remission was achieved, two patients showed a partial remission, three resulted in stable disease and three patients progressed. Natural killer and lymphokine-activated killer cell activity dramatically decreased 1 h after the beginning of rIL-2 infusion and increased at the end of treatment. A progressive increase of circulating CD8+ and HLA class II+ T cells as well as of CD8+ T cell clones, most of which displayed NK activity, was recorded following rIL-2 infusion. Present data indicate that (a) the local administration of rIL-2 coupled with systemic rIL-2 infusion may be suggested as an alternative approach for the immunotherapy of lung cancer, (b) rIL-2 induces different immunological modifications according to the route and the time of its administration and (c) rIL-2 administration increases the amount of circulating immune cells with potential antitumour activity.

In the present study T lymphocytes isolated from a metastatic lymph node (T-LNL) of a melanoma patient have been cloned. In the attempt to verify whether T-LNL may acquire in vitro functional activities in the absence of tumour-associated antigens, they were cloned utilizing allogeneic lymphocytes as feeder cells. Nineteen clones generated from T-LNL proved to be CD4+ and, among these, five were able to kill autologous and allogeneic human melanoma cells in HLA-class-II-restricted way. On the basis of their cytokine production, these CD4+ cytolytic T-LNL clones were shown to belong to the Th0 subset and three of them expersseed the Vß17 chain of the T cell receptor. These results suggest the presence of melanomaspecific but functionally inactive lymphocytes with T cell receptor oligoclonality in the lymph node environment. These specific T cells may acquire in vitro the capacity to kill autologous and allogeneic tumours without any induction by autologous melanoma cells.

The relative potency of interferon a (IFNa), interferon ß (IFNß), and interferon ? (IFN?) in inducing the expression of HLA class I antigens, as well as their capacity to counteract the inhibition induced by glucocorticoid hormones on HLA class I antigen expression, were analysed in the human melanoma cell line M14, both at membrane and at mRNA level. The data obtained indicate that (a) IFN enhance with different potency (IFN?>IFNß>IFNa) the expression of HLA class I antigens in M14 cells, (b) prednisone inhibits HLA class I antigen expresion, (c) glucocorticoid hormones, when associated with IFNa or IFN?, inhibit the HLA class I enhancement induced by IFN alone, and ffinally, (c) the association between 1 µM prednisone or 1 µM deflazacort and IFNß seems to potentiate the enhancing capacity of IFN on the expression of HLA class I molecules at the mRNA level. These findings, if confirmed, might indicate that IFN and glucocorticoid hormones are not mutually exclusive in the management of human melanoma.

CD4⁺ T cells proliferating in response to purified double-stranded deoxyribonucleic acid (dsDNA) have been recently demonstrated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Their activation was inhibited by anti-HLA class II (HLA-II) monoclonal antibodies; thus, the existence of a molecular interaction between dsDNA and HLA-II is conceivable. In this report we show that dsDNA specifically bind to HLA-II. After preincubating cells with purified dsDNA or synthetic oligonucleotides, dsDNA was detected on the cell membrane and in the lysates of HLA-II⁺ but not of isogenic HLA-II^– cell lines. We demonstrate that dsDNA binding inhibits that of a specific peptide to HLA-II. Mixed lymphocyte reaction and antigen-specific T cell proliferation were inhibited by the preincubation of stimulator cells or antigen-presenting cells with dsDNA. These results suggest the existence of a novel mechanism of down-modulation of the CD4⁺ T cell function generated by lack of stimulation due to the HLA-II presenting molecules being --bb--occupied" by dsDNA.

Soluble HLA class I antigens (sHLA-I),ß2-microglobulin (ß2-µ.) and alanine aminotransferase (ALT) serum levels have been evaluated in 16 patients affected by chronic hepatitis C treated for six months with recombinant interferon-a (rIFN-a, 3 MU three times a week). The predictor role of sHLA-I and ALT modifications with respect to the response to rIFN-a therapy was also evaluated. Six patients responded (group 1), five patients relapsed following an initial response (group 2), and five did not respond to rIFN-a treatment (group 3). The baseline serum levels of sHLA-I andß2-µ were significantly higher in all three groups of HCV-positive patients with respect to HCV-negative controls (P<0.05). A significant increase of sHLA-I serum level with respect to baseline value (P<0.001) was observed in group 1 patients after two weeks of rIFN-a treatment. sHLA-I serum level then decreased, although remaining steadily and significantly increased with respect to baseline (P values ranging from 0.05 to 0.01) in the following five months and then returned to baseline one month after the end of rIFN-a administration. No significant variations ofß2-µ serum levels were detected throughout the observation period. In group 1 patients ALT serum levels significantly decreased after two weeks of rIFN-a treatment (P<0.001) and then remained in the normal range throughout the observation period. In the other two groups of patients no relevant variations of sHLA-I andß2-µ serum levels were found during and after rIFN-a therapy. The modifications of sHLA-I serum levels discriminate, as a single marker, group 1 patients from group 2 and 3 patients after two weeks of rIFN-a treatment (P<0.003). The association of sHLA-I and ALT modifications improves the discriminant power and leads to a complete differentiation of the three groups of patients after four weeks of rIFN-a treatment (P<0.0001). If confirmed in a larger series of patients, these results will provide a useful marker to predict which patients affected by chronic hepatitis C will respond to treatment and will help to avoid their ineffective treatment with an expensive and potentially harmful drug.

Recent evidence suggests that opiates can modulate the immune responses. In particular it has been shown that β-endorphin and morphine are able to depress some T lymphocyte functions in humans. In the present study, experiments were designed to evaluate the effect of β-endorphin phytohemagglutinin-induced lymphocyte proliferation and determine the mechanism of this action. The ability of naloxone to block the effect of β-endorphin was also investigated, and the influence of β-endorphin on the expression of mononuclear cell surface antigens using the OKT3, OKT4, OKT8, anti-HLA-DR and anti-β2-microglobulin monoclonal antibodies was evaluated. Phytohemagglutinin-induced lymphocyte proliferation was significantly inhibited by β-endorphin. This effect occurred when β-endorphin was added to cells at the beginning of the culture period (30 min before, simultaneously or 30 min after phytohemagglutinin), but not when added after 48 h of incubation. The preincubation of cells with BEP for 1 h, 4 h or 24 h did not affect lymphocyte activation by phytohemagglutinin. A ten-fold excess of naloxone, added to cultures 30 min prior to β-endorphin, did not block the inhibitory effect. Incubation with β-endorphin had different effects on each surface antigen tested. The OKT8+and β2-microglobulin+cells did not show significant variations. The OKT4+cells significantly decreased, after 4 h of incubation with β-endorphin, both in mononuclear cell and in purified T lymphocyte cultures and, after 24 h, in mononuclear cell cultures only. The OKT3+cells decreased, in mononuclear cell cultures only, after 24 h β-endorphin incubation. The HLA-DR+cells significantly increased after a 24 hr incubation period with β-endorphin in mononuclear cell cultures. These results demonstrate that β-endorphin inhibits phytohemagglutinin-induced lymphocyte proliferation, enhances the expression of HLA-DR antigen in mononuclear cells and reduces the expression of T3 and T4 antigens in T lymphocytes.

ABSTRACTCovert human immunodeficiency virus (HIV) replication was ongoing during the first 3 years of aviremia in 22 patients, as determined by detection of DNA containing two long terminal repeats (2LTR DNA). Although total HIV DNA was detected in 60 2LTR DNA-negative samples, the absence of 2LTR DNA in 90% of patients following 7 to 8 years of highly active antiretroviral therapy suggests suppression of cryptic viral replication.

No relevant conflicts of interest to declare.

Anemia is one of the most prevalent clinic condition leading to a specialist medical consult. In 2014 our Internal Medicine unit started a Multidisciplinary Anemia Ambulatory (Internist, Immune-Hematologist, Hematologist) with the purpose to rapidly manage, diagnosis and treatment of anemic patients, giving a direct connection between general practitioners and hospital services. We treated and collected data on patients come to our attention in a tertiary care hospital in Genoa (Liguria), an area characterized by elderly population, which often carries more than one comorbidity with the purpose of better define the epidemiology of such a prevalent but underestimated issue. From January 1st2014 a total of 212 patients came to our attention for internist consult due to anemia: 165 female and 47 male, medium age 63,23 years (F 58,86, M 78,57, range 19-100). A precise classification of anemia was determined for 187 patients: 130 had iron deficiency anemia (IDA, 61,32%), 17 multifactorial anemia (inflammatory disorders, chronic kidney disease and combined deficiency, 8,02%), 16 combined deficiency anemia (iron and vitamins, 7,55%), 9 chronic kidney disease related anemia (4,25%), 7 anemia secondary to inflammatory chronic disorder (3,30%), 5 B12 deficiency (2,36%), 2 both folate and B12 deficiency (0,94%), 1 folate deficiency (0,47%). Twenty-five patients were not classified due to lack of data. Severity of anemia was defined according to WHO criteria: 53 patients (25%) presented mild anemia (Hb 129 - 110 g/L), 123 (58%) moderate anemia (Hb 109 - 80 g/L), 33 (15,6%) severe anemia (Hb < 80 g/L). Three patients were not anemic at the baseline evaluation. We considered comorbidities of internistic relevance, which could be worsened by anemia: cardiovascular (coronary heart disease, arrhythmias, heart failure), 30 patients; neurologic (ischemic and degenerative diseases), 19 patients; respiratory disease (COPD and asthma), 11 patients. Eleven patients had 2 comorbidities (cardiovascular and respiratory or neurological) and 3 patients had all three comorbidities. Patients were treated according to clinical practice in relation to type, severity and clinical manifestation of anemia. One hundred and thirty patients needed more than one access to ambulatory to correct anemia; data from the second access were: patient responders (normalization of Hb levels or improvement of at least 20 g/L): 78 patients; partial responders (improvement of Hb levels from 5 to 20 g/L): 34 patients; non responders: 18 patients. Fourteen patients needed at least 1 blood red cells transfusion, 12 with severe anemia and 2 with moderate anemia. A total of 93 patients needed deep diagnostic insight through specialist pathways, such as hematologic (4 patients), gastroenterologic (39 patients), gynecologic (37 patients), both gastroenterologic and gynecologic (13 patients). All patients were managed as outpatients, except for 8 patients which required hospitalization due to severity of clinical findings: 4 patients were hospitalized in Internal Medicine ward, 1 patients in Gynecology and 3 patients needed access through Emergency Care Unit. Among IDA patients, 92 were treated with intravenous iron supplement: 32 with sodium ferric gluconate (SFG) (medium 16,68 vials, range 8-43) and 50 with ferric carboxymaltose (FC) (medium 1,04 vials). Nine patients treated with SFG experienced allergic reaction, so they were switched to FC. Patients treated with SFG were successfully treated for 69,56% and 26,08% responded partially. One patient treated with FC experienced allergic reaction, so he was switched to oral therapy. FC patients fully responded in 76% and 22% were partial responders. These preliminary data shows that Multidisciplinary Anemia Ambulatory and its diagnostic-therapeutic path, with the involvement of different Specialists and Operative Unit, resulted in an improvement of the coordination and continuity of care, reducing sanitary cost in terms of hospitalization, drugs rationalization and quality of life for patients. More data will derive from the newborn Anemia Regional Register, which will lead to a better comprehension of the real size of anemia in our local epidemiology, in which health derived costs are rising together with ageing of the comorbid population which often needs longitudinal assistance, coordination and continuity of care.