[Hematological diagnosis in the corneal consultation]. / Hämatologische Diagnose in der Hornhautsprechstunde.

This case report describes a diagnostic chain in a patient with atypical bilateral corneal opacity, which led to the diagnosis of a hematological disorder. The patient's medical history, clinical appearance and findings in confocal microscopy gave rise to the suspicion of a paraproteinemic keratopathy. The hematological laboratory diagnostics revealed a monoclonal gammopathy of the IgG kappa type. The bone marrow puncture led to the diagnosis of a lymphoplasmacytic lymphoma, which belongs to the group of B­cell non-Hodgkin's lymphomas. This case demonstrates that paraproteinemic keratopathy can be associated with potentially life-threatening hematological diseases.

A randomized phase III study of carfilzomib vs low-dose corticosteroids with optional cyclophosphamide in relapsed and refractory multiple myeloma (FOCUS).

This randomized, phase III, open-label, multicenter study compared carfilzomib monotherapy against low-dose corticosteroids and optional cyclophosphamide in relapsed and refractory multiple myeloma (RRMM). Relapsed and refractory multiple myeloma patients were randomized (1:1) to receive carfilzomib (10-min intravenous infusion; 20 mg/m2 on days 1 and 2 of cycle 1; 27 mg/m2 thereafter) or a control regimen of low-dose corticosteroids (84 mg of dexamethasone or equivalent corticosteroid) with optional cyclophosphamide (1400 mg) for 28-day cycles. The primary endpoint was overall survival (OS). Three-hundred and fifteen patients were randomized to carfilzomib (n=157) or control (n=158). Both groups had a median of five prior regimens. In the control group, 95% of patients received cyclophosphamide. Median OS was 10.2 (95% confidence interval (CI) 8.4-14.4) vs 10.0 months (95% CI 7.7-12.0) with carfilzomib vs control (hazard ratio=0.975; 95% CI 0.760-1.249; P=0.4172). Progression-free survival was similar between groups; overall response rate was higher with carfilzomib (19.1 vs 11.4%). The most common grade ⩾3 adverse events were anemia (25.5 vs 30.7%), thrombocytopenia (24.2 vs 22.2%) and neutropenia (7.6 vs 12.4%) with carfilzomib vs control. Median OS for single-agent carfilzomib was similar to that for an active doublet control regimen in heavily pretreated RRMM patients.

Malnutrition in Healthy Individuals Results in Increased Mixed Cytokine Profiles, Altered Neutrophil Subsets and Function.

Malnutrition is commonly associated with increased infectious disease susceptibility and severity. Whereas malnutrition might enhance the incidence of disease as well as its severity, active infection can in turn exacerbate malnutrition. Therefore, in a malnourished individual suffering from a severe infection, it is not possible to determine the contribution of the pre-existing malnutrition and/or the infection itself to increased disease severity. In the current study we focussed on two groups of malnourished, but otherwise healthy individuals: moderately malnourished (BMI: 18.4-16.5) and severely malnourished (BMI <16.5) and compared several immune parameters with those of individuals with a normal BMI (≥18.5). Our results show a similar haematological profile in all three groups, as well as a similar ratio of CD4+ and CD8+ T cells. We found significant correlations between low BMI and increased levels of T helper (Th) 1 (Interferon (IFN)-γ, (interleukin (IL)-2, IL-12), Th2 (IL-4, IL-5, IL-13), as well as IL-10, IL-33 and tumor necrosis factor-α, but not IL-8 or C reactive protein. The activities of arginase, an enzyme associated with immunosuppression, were similar in plasma, peripheral blood mononuclear cells (PBMC) and neutrophils from all groups and no differences in the expression levels of CD3ζ, a marker of T cell activation, were observed in CD4+ and CD8+T cells. Furthermore, whereas the capacity of neutrophils from the malnourished groups to phagocytose particles was not impaired, their capacity to produce reactive oxygen species was impaired. Finally we evaluated the frequency of a subpopulation of low-density neutrophils and show that they are significantly increased in the malnourished individuals. These differences were more pronounced in the severely malnourished group. In summary, our results show that even in the absence of apparent infections, healthy malnourished individuals display dysfunctional immune responses that might contribute to increased susceptibility and severity to infectious diseases.

Phase III trial of bortezomib, cyclophosphamide and dexamethasone (VCD) versus bortezomib, doxorubicin and dexamethasone (PAd) in newly diagnosed myeloma.

We aimed at demonstrating non-inferiority of bortezomib/cyclophosphamide/dexamethasone (VCD) compared to bortezomib/doxorubicin/dexamethasone (PAd) induction therapy with respect to very good partial response rates or better (⩾VGPR) in 504 newly diagnosed, transplant-eligible multiple myeloma patients. VCD was found to be non-inferior to PAd with respect to ⩾VGPR rates (37.0 versus 34.3%, P=0.001). The rates of progressive disease (PD) were 0.4% (VCD) versus 4.8% (PAd; P=0.003). In the PAd arm, 11 of 12 patients with PD had either renal impairment (creatinine ⩾2 mg/dl) at diagnosis or the cytogenetic abnormality gain 1q21, whereas no PD was observed in these subgroups in the VCD arm. Leukocytopenia/neutropenia (⩾3°) occurred more frequently in the VCD arm (35.2% versus 11.3%, P<0.001). Neuropathy rates (⩾2°) were higher in the PAd group (14.9 versus 8.4%, P=0.03). Serious adverse events, both overall and those related to thromboembolic events, were higher in the PAd group (32.7 versus 24.0%, P=0.04 and 2.8 versus 0.4%, P=0.04). Stem cell collection was not impeded by VCD. VCD is as effective as PAd in terms of achieving ⩾VGPR rates with fewer PD and has a favorable toxicity profile. Therefore, VCD is preferable to PAd as induction therapy.

Antiretroviral therapy abrogates association between arginase activity and HIV disease severity.

Arginase-induced L-arginine deprivation is emerging as a key mechanism for the downregulation of immune responses. We hypothesised that arginase activity increases with disease severity in HIV-seropositive patients. Our results show that peripheral blood mononuclear cells (PBMCs) from 23 HIV-seropositive patients with low CD4(+) T cell counts (≤350 cells/µl) expressed significantly more arginase compared with 21 patients with high CD4(+) T cell counts. Furthermore, we found a significant association between the two principal prognostic markers used to monitor HIV disease (CD4(+) T cell count and plasma viral load) and PBMC arginase activity in antiretroviral therapy naïve patients but not in patients undergoing therapy.

Increased level of arginase activity correlates with disease severity in HIV-seropositive patients.

Infection with human immunodeficiency virus (HIV) results in a chronic infection that progressively impairs the immune system. Although depletion of CD4(+) T cells is frequently used to explain immunosuppression, chronicity of infection and progressive loss of CD4(+) T cells are not sufficient to fully account for immune dysregulation. Arginase-induced l-arginine deprivation is emerging as a key mechanism for the down-regulation of immune responses. Here, we hypothesized that the level of arginase activity increases with disease severity in HIV-seropositive patients. We determined the levels of arginase activity in peripheral blood mononuclear cells from HIV-seropositive patients and uninfected control participants. Our results show that peripheral blood mononuclear cells from HIV-seropositive patients with low CD4(+) T cell counts expressed statistically significantly higher levels of arginase activity, compared with patients with high CD4(+) T cell counts or uninfected control participants. Furthermore, we found a statistically significant correlation between high level of arginase activity and high viral load in HIV-seropositive patients.

Differential impact of L-arginine deprivation on the activation and effector functions of T cells and macrophages.

The metabolism of the amino acid L-arginine is emerging as a crucial mechanism for the regulation of immune responses. Here, we characterized the impact of L-arginine deprivation on T cell and macrophage (MPhi) effector functions: We show that whereas L-arginine is required unconditionally for T cell activation, MPhi can up-regulate activation markers and produce cytokines and chemokines in the absence of L-arginine. Furthermore, we show that L-arginine deprivation does not affect the capacity of activated MPhi to up-regulate L-arginine-metabolizing enzymes such as inducible NO synthase and arginase 1. Thus, our results show that to exert their effector functions, T cells and MPhi have different requirements for L-arginine.

The initial phase of graft-versus-host disease is associated with a decrease of CD4+CD25+ regulatory T cells in the peripheral blood of patients after allogeneic stem cell transplantation.

The mechanisms that induce and control the alloimmune inflammation of graft-versus-host disease (GvHD) after allogeneic stem cell transplantation (allo-SCT) are still incompletely understood. In the murine system, GvHD can be suppressed by CD4(+)CD25(+) regulatory T cells (TREG), which are generally involved in the suppression of inflammatory reactions. A disruption of the homeostasis between TREG and conventional T cells might therefore be associated with the inflammatory reactions of GvHD. We repetitively measured the frequency of TREG in the peripheral blood of 29 patients within the first 71-373 days after allo-SCT and correlated the results with the clinical course. We demonstrate that the initial phase of GvHD is associated with a significant reduction of TREG in the peripheral blood, while at later stages and during intensified immunosuppressive therapy, increased numbers of TREG appear. These results might indicate a pathogenic role for reduced numbers of TREG in the induction of human GvHD.

Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells.

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMPhi) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMPhi. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMPhi and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMPhi, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.

Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety.

In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of [1'-14C]riboflavin. The vitamin B12 isolated was 14C-labeled. It was shown by chemical degradation that the 14C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety. This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S. typhimurium.

Murine macrophages secrete interferon gamma upon combined stimulation with interleukin (IL)-12 and IL-18: A novel pathway of autocrine macrophage activation.

Interferon (IFN)-gamma, a key immunoregulatory cytokine, has been thought to be produced solely by activated T cells and natural killer cells. In this study, we show that murine bone marrow- derived macrophages (BMMPhi) secrete large amounts of IFN-gamma upon appropriate stimulation. Although interleukin (IL)-12 and IL-18 alone induce low levels of IFN-gamma mRNA transcripts, the combined stimulation of BMMPhi with both cytokines leads to the efficient production of IFN-gamma protein. The macrophage-derived IFN-gamma is biologically active as shown by induction of inducible nitric oxide synthase as well as upregulation of CD40 in macrophages. Our findings uncover a novel pathway of autocrine macrophage activation by demonstrating that the macrophage is not only a key cell type responding to IFN-gamma but also a potent IFN-gamma-producing cell.

Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4+ T cells correlates with Th1/Th2 phenotype.

Activated murine macrophages metabolize L-arginine via two main pathways that are catalyzed by the inducible enzymes nitric oxide synthase (iNOS) and arginase. We have previously shown that CD4+ T cell-derived cytokines regulate a competitive balance in the expression of both enzymes in macrophages; Thl-type cytokines induce iNOS while they inhibit arginase, whereas the reverse is the case for Th2-type cytokines. Here we addressed the regulation of both metabolic pathways by CD4+ T cells directly. Macrophages were used as APCs for established Th1 and Th2 T cell clones as well as for in vitro polarized Th1 or Th2 T cells of transgenic mice bearing an MHC class II-restricted TCR. Both systems revealed a similar dichotomy in the macrophages; Th1 T cells led to an exclusive induction of iNOS, whereas Th2 T cells up-regulated arginase without inducing iNOS. Arginase levels induced by Th2 T cells far exceeded those inducible by individual Th2 cytokines. Similarly, high arginase levels could be induced by supernatants of Th2 cells stimulated in various ways. Ab blocking experiments revealed the critical importance of IL-4 and IL-10 for arginase up-regulation. Finally, strong synergistic effects between IL-4/IL-13 and IL-10 were observed, sufficient to account for the extraordinarily high arginase activity induced by Th2 cells. Our results suggest that the iNOS/arginase balance in macrophages is competitively regulated in the context of Th1- vs Th2-driven immune reactions, most likely by cytokines without the requirement for direct cell interaction.

Identification of the porcine intestinal accessory factor that enables DNA sequence recognition by vitamin D receptor.

The nuclear accessory protein in porcine intestinal nuclear extracts that activates the binding of the vitamin D receptor to its vitamin D response elements has been highly purified. It contains a protein that binds 9-cis-[3H]retinoic acid, was detected on immunoblots with an anti-retinoid X receptor (RXR) peptide antibody, and supports the binding of retinoic acid receptor gamma to the retinoic acid receptor beta gene response element. Most important, the two specific complexes formed by porcine nuclear extract with the vitamin D response elements from either the osteocalcin gene or the rat 24-hydroxylase gene are shifted to a larger complex by both an anti-vitamin D receptor antibody and an anti-RXR antibody, leaving no doubt that in vivo the nuclear accessory factor for the vitamin D receptor in the intestine is an RXR protein.

Biosynthesis of vitamin B12 in anaerobic bacteria. Experiments with Eubacterium limosum on the incorporation of D-[1-13C]erythrose and [13C]formate into the 5,6-dimethylbenzimidazole moiety.

Experiments on the incorporation of erythrose and formate into the 5,6-dimethylbenzimidazole moiety of vitamin B12 are described. In one experiment, a 1:1 mixture of D-[1-13C]erythrose and D-[1-13C]threose was added to a Eubacterium limosum fermentation. The vitamin B12 formed was methylated at N3 of its 5,6-dimethylbenzimidazole part and degraded to 1,5,6-trimethylbenzimidazole. The 13C-NMR spectrum of this compound exhibited a single prominent signal at 109.5 ppm due to 13C labeling in C7. This shows that C1 of erythrose or threose was originally incorporated exclusively into C4 of the 5,6-dimethylbenzimidazole moiety of vitamin B12. In another experiment, sodium [13C]formate was added to a culture of E. limosum. The vitamin B12 isolated was transformed into 1,5,6-trimethylbenzimidazole as before. The 13C-NMR spectrum also showed one prominent signal at 142.8 ppm, evoked by 13C at C2. These results demonstrate that erythrose is incorporated into the base part of vitamin B12 regiospecifically and that formate is the precursor of the C2.