Plasmacytoid dendritic cells of melanoma patients present exogenous proteins to CD4+ T cells after Fc gamma RII-mediated uptake.

Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immune responses by producing type I interferons. Although human pDCs can induce T cell responses upon viral infection, it remains unclear if pDCs can present exogenous antigens. Here, we show that human pDCs exploit FcgammaRII (CD32) to internalize antigen-antibody complexes, resulting in the presentation of exogenous antigen to T cells. pDCs isolated from melanoma patients vaccinated with autologous monocyte-derived peptide- and keyhold limpet hemocyanin (KLH)-loaded dendritic cells, but not from nonvaccinated patients or patients that lack a humoral response against KLH, were able to stimulate KLH-specific T cell proliferation. Interestingly, we observed that internalization of KLH by pDCs depended on the presence of serum from vaccinated patients that developed an anti-KLH antibody response. Anti-CD32 antibodies inhibited antigen uptake and presentation, demonstrating that circulating anti-KLH antibodies binding to CD32 mediate KLH internalization. We conclude that CD32 is an antigen uptake receptor on pDCs and that antigen presentation by pDCs is of particular relevance when circulating antibodies are present. Antigen presentation by pDCs may thus modulate the strength and quality of the secondary phase of an immune response.

Intratumoral rhIL-12 administration in head and neck squamous cell carcinoma patients induces B cell activation.

The objectives of this study were to investigate the effects of intratumorally (i.t.) administered recombinant human interleukin-12 (rhIL-12) on the distribution and function of B cells in the primary tumors, the locoregional lymph nodes and peripheral blood of head and neck squamous cell carcinoma (HNSCC) patients. The initial characterization of the patients participating in the phase Ib and phase II studies has previously been reported. After rhIL-12 treatment, fewer secondary follicles with a broader outer region of the mantle zones and an increase in interfollicular B-blasts were seen in the enlarged lymph nodes compared with control HNSCC patients. The size of the germinal center (GC) was diminished, partly due to a decrease in the number of CD57+ GC cells that have been associated with immune suppression. These changes did not correlate with signs of apoptosis or CXCR5 expression by B cells. Strikingly, in 3 out of 4 IL-12 treated patients, increased IFN-gamma mRNA expression by B cells was detected. In addition, a highly significant IgG subclass switch was seen in the plasma with more IgG1, less IgG2 and more IgG4, indicating a switch to T helper 1 phenotype. Finally, peritumoral B cell infiltration was a positive prognostic sign for overall survival in the 30 HNSCC patients investigated, irrespective of IL-12 treatment. In conclusion, these data indicate that after i.t. IL-12 treatment in HNSCC, significant activation of the B cell and the B cell compartment occurred and that the presence of tumor infiltrating B cells correlated with overall survival of HNSCC patients.

Antibodies against the CUB1-2 domains of ADAMTS13 in a patient with benign monoclonal gammopathy: no causal relationship.

We present a patient with a history of benign monoclonal gammopathy, who developed thrombotic thrombocytopenic purpura (TTP), initially presenting as bilateral serous retinal detachment. Plasma of the patient contained high titers of anti ADAMTS13 antibodies that were directed towards the disintegrin/TSR1/cysteine-rich/spacer and CUB1-2 domains. ADAMTS13 activity was undetectable. Total IgG purified from plasma of the patient partially inhibited ADAMTS13 activity. In contrast, the isolated M-protein did neither bind to, nor inhibit activity of ADAMTS13. We conclude that in this patient the monoclonal gammopathy and TTP co-existed as distinct pathological entities.

Complement activation in experimental human malaria infection.

The objective of this study was to investigate complement activation in uncomplicated, early phases of human malaria. Fifteen healthy volunteers were experimentally infected with Plasmodium falciparum malaria. Parasitemia and complement activation products were assessed. During blood stage parasitemia, volunteers showed a significant increase in soluble terminal complement complex (TCC) formation. After start of a curative regimen of artemether/lumefantrine, TCC further increased due to activation of both the classical and the alternative pathway. In-vitro studies confirmed activation of complement by parasite cultures. We thus detected an increase in complement activation in volunteers with experimentally induced malaria, even before parasitemia could be detected microscopically. This significant increase in complement activation occurred despite the possible control of TCC formation by complement regulatory proteins on erythrocytes and the extremely low levels of parasitemia. Treatment with artemether/lumefantrine was followed by classical and alternative pathway complement activation, without evidence for mannan-binding-lectin-mediated complement activation.

Cerebrospinal fluid IgM index correlates with cranial MRI lesion load in patients with multiple sclerosis.

In multiple sclerosis intrathecal IgM synthesis correlates with an unfavourable disease course. Whether this reflects a pathogenic role of IgM, possibly in conjunction with complement, is a matter of debate. In a cross-sectional study we measured intrathecal synthesis of IgM and the complement component C3, and on cranial MRI lesion load and central brain atrophy in clinically active patients, 17 relapsing-remitting, 16 secondary progressive. Correlative analysis showed that in relapsing-remitting patients CSF IgM index correlated with cranial MRI T2 and T1 lesion load, and central brain atrophy; and the C3 index correlated with T2 lesion load. In secondary progressive patients CSF IgM index correlated with periventricular T2 lesion load. Our data are in favour of a pathogenic role of IgM in multiple sclerosis.

The fractional excretion of soluble interleukin-2 receptor-alpha is an excellent predictor of the interleukin-2 receptor-alpha status after treatment with daclizumab.

BACKGROUND: Daclizumab is a humanized monoclonal antibody against the alpha-chain of the interleukin (IL)-2 receptor (R). The authors previously have shown that the urinary excretion of soluble (s) IL-2Ralpha is dependent on the presence of daclizumab in serum. The authors investigated whether the IL-2Ralpha status, as assessed by flow cytometric analysis, is reflected by the concentration of sIL-2Ralpha in the urine and serum. METHODS: Two hundred seventy-two measurements were performed in 46 renal transplant recipients who were treated with daclizumab in combination with tacrolimus and mycophenolate mofetil. Soluble IL-2Ralpha was measured in urine and serum with Immulite IL-2R, a solid-phase enzyme-linked immunosorbent assay. Complete blockade of the IL-2Ralpha was defined as the presence of less than 5% IL-2Ralpha+ lymphocytes in the CD3+ population. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the performance of serum and urine sIL-2Ralpha in predicting IL-2Ralpha blockade. RESULTS: The calculated fractional excretion of sIL-2Ralpha proved to be an excellent predictor of the blockade of IL-2Ralpha (ROC analysis area under the curve, 0.95+/-0.01). A calculated fractional excretion of sIL-2Ralpha lower than 0.5% had a specificity of 100% and a sensitivity of 75% for the assessment of blockade of IL-2Ralpha. CONCLUSIONS: Blockade of IL-2Ralpha after treatment with daclizumab can reliably be assessed by calculation of the fractional excretion of sIL-2Ralpha. This method is easier to use compared with flow cytometric analysis of IL-2Ralpha+ lymphocytes.

The complement system is activated in a biphasic pattern after coronary artery bypass grafting.

BACKGROUND: The complement system is a key component in the inflammatory response after coronary artery bypass grafting (CABG). The routes of complement activation and deactivation after cardiac surgery are not clear. The aim of this study was to analyze routes of complement activation after uncomplicated CABG. METHODS: Complement components and activation products were measured in 20 nondiabetic adult patients undergoing elective CABG at several times postoperatively starting at admission to the intensive care unit. RESULTS: Complement activation after uncomplicated CABG showed a biphasic pattern. In the first 8 hours after admission to the intensive care unit, complement activation was initiated by the classical lectin pathway and augmented by the alternative pathway. Ultimately, this resulted in terminal pathway activation and formation of terminal complement complex. In the second phase, starting at 8 hours after the operation, complement was still activated by the classical lectin pathway, but there was no augmentation by the alternative pathway and no terminal complement complex formation. This implies that during this second stage, inhibitory mechanisms beyond C3b are engaged. CONCLUSIONS: Complement activation after cardiac surgery is regulated in a complex biphasic way, with additional inhibitory mechanisms engaged from 8 hours postoperatively onward.

Dendritic cell vaccination in combination with anti-CD25 monoclonal antibody treatment: a phase I/II study in metastatic melanoma patients.

PURPOSE: The success of cancer immunotherapy depends on the balance between effector T cells and suppressive immune regulatory mechanisms within the tumor microenvironment. In this study we investigated whether transient monoclonal antibody-mediated depletion of CD25(high) regulatory T cells (Treg) is capable of enhancing the immunostimulatory efficacy of dendritic cell vaccines. EXPERIMENTAL DESIGN: Thirty HLA-A2.1(+) metastatic melanoma patients were vaccinated with mature dendritic cells pulsed with tumor peptide and keyhole limpet hemocyanin (KLH). Half of the patients were pretreated with daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor α-chain (CD25), either four or eight days before dendritic cell vaccinations. Clinical and immunologic parameters were determined. RESULTS: Daclizumab efficiently depleted all CD25(high) immune cells, including CD4(+)FoxP3(+)CD25(high) cells, from the peripheral blood within four days of administration. Thirty days after administration, daclizumab was cleared from the circulation and all CD25(+) cells reappeared. The presence of daclizumab during dendritic cell vaccinations prevented the induction of specific antibodies in vivo but not the presence of antigen-specific T cells. Daclizumab, however, did prevent these CD25(+) T cells from acquiring effector functions. Consequently, significantly less patients pretreated with daclizumab developed functional, vaccine-specific effector T cells and antibodies compared with controls. Daclizumab pretreatment had no significant effect on progression-free survival compared with the control group. CONCLUSIONS: Although daclizumab depleted the CD4(+)FoxP3(+)CD25(high) Tregs from the peripheral circulation, it did not enhance the efficacy of the dendritic cell vaccine. Residual daclizumab functionally suppressed de novo induced CD25(+) effector cells during dendritic cell vaccinations. Our results indicate that for immunotherapeutic benefit of transient Treg depletion, timing and dosing as well as Treg specificity are extremely important.

Autoantibody standardization in The Netherlands.

Several initiatives have been undertaken, independent of the European Autoantibody Standardization Initiative (EASI), to standardize autoantibodies in The Netherlands. The Dutch EASI team has made an inventory of which initiatives on autoantibody standardization are already available and what future plans for autoantibody standardization exist. This inventory will subsequently be used to define what may be addressed by the Dutch EASI team. The Diagnostic Compass, initiated by the association of Dutch health insurance companies, describes methods and relevance of laboratory tests, including autoantibody tests. Recently, this initiative has been taken over by an independent publisher. There is also a national organization involved in developing guidelines in medicine, including guidelines for autoantibody testing. In addition, there is a national foundation for quality assessment in clinical laboratories (SKML). The quality assessment includes a wide array of autoantibodies. Samples are collected and thoroughly investigated by reference laboratories. Interpretation of results and advice to clinicians are part of the program. Feedback on the results of this proficiency testing is given in reports and during meetings to discuss trends, technical issues, and new developments. The last initiative that we discuss is the foundation Referentie Laboratorium Reuma Serologie (RELARES), which was founded to standardize serology in rheumatic diseases by preparing standard sera. Recently, RELARES has been combined with SKML. A new SKML working group, Standardization Autoimmune Serology, has been initiated to continue the work of RELARES. When comparing the already available Dutch initiatives to the international EASI goals, there appears to be a lack of harmonization in testing algorithms, and this issue is the most important topic to be addressed in the near future.

A missense mutation in factor I (IF) predisposes to atypical haemolytic uraemic syndrome.

A genetic predisposition involving complement regulatory genes has become evident in some patients with atypical HUS. In this paper, a patient with a heterozygous missense mutation in factor I (IF) is described. Although the serum level of IF was normal, a mild functional defect in the alternative pathway of complement could be demonstrated in the affected members of the family. After an episode of atypical HUS, chronic renal insufficiency started at the age of 15 months. Recurrence of HUS, with loss of the renal transplant, occurred twice in this patient. The recurrence of HUS in the graft was not reflected by haematological abnormalities (haemolysis, thrombocytopenia). One additional transplant was lost due to arterial thrombosis of the renal artery. This report confirms the gloomy outcome of renal transplants in patients with an IF deficiency. New therapies should be evaluated in these patients.

Urinary excretion of beta2-microglobulin and IgG predict prognosis in idiopathic membranous nephropathy: a validation study.

An accurate prediction of the prognosis of patients with idiopathic membranous nephropathy (iMN) should allow restriction of immunosuppressive treatment to patients who are at highest risk for ESRD. On the basis of retrospective studies, it has previously been suggested that the urinary excretions of beta2-microglobulin (Ubeta2m) and IgG (UIgG) are useful predictors of renal insufficiency in patients with iMN. The threshold values of 0.5 micro/min (Ubeta2m) and 250 mg/24 h (UIgG) have been validated in a new and larger patient cohort. From 1995 onward, 57 patients with iMN (38 men, 19 women; age 48 +/- 16 yr), a nephrotic syndrome, and a serum creatinine level 50%. Mean (+/-SD) follow-up was 53 +/- 23 mo. Thus far, 25 (44%) of the patients have reached the end point renal death. Multivariate analysis confirmed Ubeta2m as the strongest independent predictor for the development of renal insufficiency. Sensitivity and specificity were 88 and 91%, respectively, for Ubeta2m, and both were 88% for UIgG. When the excretions of both proteins were combined, specificity improved to 97%. It is concluded that the present data validate the accuracy of Ubeta2m and of UIgG in predicting renal outcome in patients with iMN. These markers can be used to guide decisions on the start of immunosuppressive treatment.

Serum ferritin levels are increased in patients with glomerular diseases and proteinuria.

BACKGROUND: Ferritin is a high molecular weight protein which reflects body iron stores, but may also rise in the case of an acute phase response. Recently, ferritin has been identified as a predictive factor in the development and progression of atherosclerosis. This is the first report on serum ferritin levels in patients with proteinuria. METHODS: We have analysed the data of 142 male patients with a glomerular disease, and proteinuria exceeding 1 g/day. In all patients, we measured various parameters related to proteinuria, serum ferritin and serum iron. Serum beta2-microglobulin and the Modification of Diet in Renal Disease (MDRD) equation were used as measures of the glomerular filtration rate (GFR). RESULTS: Mean age (+/-SD) was 46+/-15 years, MDRD-GFR 57+/-25 ml/min/1.73 m2 and median proteinuria 8.0 g/day [interquartile range (IQR) 3.6-13]. Serum albumin (29+/-9 g/l) and transferrin levels (1.7+/-0.5 g/l) were low, and cholesterol levels were elevated (median 7.3, IQR 5.9-9.5 mmol/l). Median serum ferritin was 148 microg/l (IQR 89-282), and exceeded 280 microg/l, the upper limit of normal, in 36 patients (25%). Elevated serum ferritin levels could not be explained by an acute phase response as determined by C-reactive protein, or haemochromatosis (DNA analysis). Regression analysis showed an independent relationship between ferritin levels and serum cholesterol, GFR and serum transferrin. CONCLUSIONS: Serum ferritin levels are elevated in patients with overt proteinuria. The independent negative relationship between serum ferritin and transferrin points to a specific process and suggests that increased production of ferritin may compensate for the loss of the iron-binding protein transferrin, thus reducing the amount of free iron. Further studies are needed to elucidate the role of ferritin in patients with proteinuria, especially because of the suggested association between ferritin and atherosclerosis.

Decreased renal excretion of soluble interleukin-2 receptor alpha after treatment with daclizumab.

BACKGROUND: Daclizumab (+/-150 kD), a humanized monoclonal antibody (mAb) against the alpha-chain of the membrane-bound interleukin-2 (IL-2) receptor (IL-2R) also binds soluble interleukin-2R alpha (sIL-2R alpha; +/-45 kD), and thus may influence the glomerular filtration of sIL-2R alpha. METHODS: We have studied the influence of daclizumab on the renal excretion of sIL-2R alpha in 38 recipients of a renal transplant (32 treated with daclizumab and six controls). sIL-2R alpha was measured every 2 weeks after transplantation in serum and urine with Immulite IL-2R, a solid-phase enzyme-linked immunosorbent assay (ELISA). RESULTS: In the control population, the fractional excretion of sIL-2R alpha was relatively constant with a median value of 1.7%+/- 0.5%. In daclizumab-treated patients, sIL-2R alpha was not detectable in the urine immediately after the administration of daclizumab. sIL-2R alpha became detectable in the urine at a mean of 8 +/- 3 weeks after transplantation. In additional experiments, serum compounds were separated by size-exclusion chromatography and sIL-2R alpha was measured in the collected fractions. In the control patients, sIL-2R alpha was only present in the low-molecular-weight fractions of serum. In contrast, in daclizumab-treated patients evaluated several weeks after transplantation, sIL-2R alpha was merely detected in the high-molecular-weight fractions of serum. During follow-up there was a relative shift of sIL-2R alpha from the high- to the low-molecular-weight fractions and this coincided with normalization of sIL-2R alpha excretion. CONCLUSION: Daclizumab inhibits the renal excretion of sIL-2R alpha by the formation of a complex with sIL-2R alpha in serum, which is too large for glomerular filtration. Measurement of urinary sIL-2R alpha may provide information on the concentration of anti-IL-2R alpha mAb in serum.