Detection of nickel and palladium contact hypersensitivity by a flow cytometric lymphocyte proliferation test.

We established a flow cytometric lymphocyte proliferation test (LPT) for the detection of nickel (Ni) and palladium (Pd) sensitization. Eighty-one consecutive patients with an indication for patch test (PT) were tested by LPT with Ni (NiSO4 ) and Pd (Na2 PdCl4 and PdCl2 ) salts. The imprecision of the LPT was low (coefficient of variation 7.2%). Using PT as a diagnostic reference, the sensitivity and specificity of LPT were 74.4% and 80% for NiSO4 , 74.4% and 78.3% for Na2 PdCl4 , and 57.2% and 85.4% for PdCl2 , respectively. For both Ni and Pd, the likelihood ratio for a positive PT markedly increased with increasing LPT value. With medical history as a reference, the sensitivity and specificity were 40.6% and 82.1% for LPT and 59.4% and 89.7% for PT, respectively. Combination of LPT and PT resulted in a higher specificity of 95%, albeit lower sensitivity of 34.4%. In conclusion, flow cytometric LPT represents a reliable and useful method for the detection of Ni and Pd sensitization. LPT values correlate with PT results and, when used in combination with PT, increase test specificity.

Skin prick test and basophil reactivity to cetuximab in patients with IgE to alpha-gal and allergy to red meat.

Severe hypersensitivity reactions to red meat with delay of several hours in patients with IgE to alpha-gal (galactose-alpha-1,3-galactose) have been reported. The diagnosis of meat allergy is difficult, because of the limited sensitivity of skin prick tests and specific IgE tests to meat extracts. These circumstances have been explained by the delayed expression of alpha-gal due to digestive processes. Because of the low sensitivity of skin prick tests to meat, we studied the possibility to perform skin prick tests with cetuximab, which carries the alpha-gal epitope. Skin prick and intradermal tests with cetuximab were clearly positive in 2 of 2 patients. As a further diagnostic step, we performed basophil activation tests with cetuximab. Skin prick tests and basophil activation test using cetuximab may be a more sensitive alternative in patients with an assumed allergy to meat.

Efficient mobilization of PBSC with vinorelbine/G-CSF in patients with malignant lymphoma.

High-dose chemotherapy (HDT) and hematopoietic SCT are effective in patients with relapsing or refractory malignant lymphoma. Collection of sufficient numbers of stem cells is a prerequisite for such a therapy. In a pilot trial, we evaluated the feasibility of stem cell mobilization with vinorelbine/G-CSF in patients with lymphoma, a regimen allowing precise timing and harvesting of sufficient stem cells in myeloma patients. Forty-five patients with lymphoma received vinorelbine 35 mg/m(2) i.v. on day 1 and G-CSF 10 microg/kg/day s.c., divided in two daily doses from day 4 until collection. Stem cell collection was successfully performed in 43 patients (96%) with a median of 3.6 x 10(6) CD34(+) cells/kg (range: 1.4-16) in the collected product. In 28 patients (62%), the first stem cell apheresis was performed on day 8, and for 28 patients a sufficient stem cell yield was reached with one apheresis only. All 43 patients underwent high-dose chemotherapy with BEAM and auto-SCT with hematological recovery on time and without unexpected toxicity. In conclusion, vinorelbine/G-CSF allows accurate timing and safe harvesting of sufficient stem cells in patients with malignant lymphoma.

[Paroxysmal nocturnal hemoglobinuria--consequences of a missing anchor]. / Paroxysmale nächtliche Hämoglobinurie--Folgen eines fehlenden Ankers.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder characterized by the clonal expansion and differentiation of a multi-potent hematopoietic stem cell carrying a somatic mutation in the X-linked PIG-A gene. As a consequence of this mutation, glycosylphosphatidylinositol (GPI)-anchored proteins are lacking on the surface of blood cells derived from the mutated stem cell. This may result clinically in hemolytic anemia and a tendency for venous thrombosis and serious infection. Bone marrow failure is a frequently observed phenomenon associated with PNH. Reliable diagnosis of PNH is currently best achieved by flow cytometric analysis of GPI-anchored proteins on peripheral blood cells. Both the clinically relevant size of the PNH clone and type of GPI deficiency (complete or partial) can be reproducibly determined by this method. Most patients will benefit from supportive measures, albeit that allogeneic hematopoietic stem cell transplantation is currently considered the only potentially curative therapy. The development of a new therapeutic monoclonal antibody that reduces intravascular hemolysis and progress in diagnostic flow cytometry using a new GPI-specific marker may provide further benefit for PNH patients in the future.

Twelve-year follow-up after discontinuation of preseasonal grass pollen immunotherapy in childhood.

BACKGROUND: In a previous controlled study, we demonstrated that preseasonal grass pollen immunotherapy for 3 years was effective in children. Moreover, a significant clinical benefit could still be observed 6 years after discontinuation of specific immunotherapy (SIT). In the current study, we examined the same group of patients again to investigate whether there is a prolonged benefit 12 years after SIT is stopped. METHODS: Twenty-two patients with previous SIT (from 1989 through 1991) or standardized seasonal pharmacotherapy only were prospectively followed during the grass pollen season of 2003. Primary end points were symptom score, medication use, and combined symptom and medication score. In addition, skin prick test reactivity, development of new sensitizations, and prevalence of seasonal asthma were evaluated. RESULTS: Total hay fever symptom score (P < 0.03), use of medication (P < 0.05), and combined symptom and medication score (P < 0.03) remained lower in patients with previous SIT when compared with the control group. Decreased immediate skin response to grass pollen returned 12 years after cessation of SIT. The percentage of new sensitization, however, continued to be significantly smaller in patients with previous SIT (58%) compared with the controls (100%, P < 0.05). There was a tendency for lower prevalence of seasonal asthma in the post-SIT group (P = 0.08). CONCLUSION: This prospective controlled prolonged follow-up study demonstrates the ongoing clinical benefit 12 years after discontinuation of SIT. Furthermore, the reduction in onset of new sensitization, which was found 6 years after discontinuation of SIT, is sustained 6 years later.

[Genetic analysis and immunophenotyping in the diagnosis of hematological disease]. / Genetische Analysen und Immunphänotypisierung in der Diagnostik hämatologischer Erkrankungen.

The developments in the fields of genetics and immunology and the application of these informations have significant consequences for the diagnosis of hematological diseases. The present article gives an introduction into the principles of several modern diagnostic techniques, which are applied in the diagnosis of hematological diseases. In addition, it summarizes the application of these techniques in the diagnosis of several acquired and inherited diseases. The most important method of immunophenotyping is FACS (fluorescence activated cell sorting) analysis, which is based on the automated recognition of fluorescently labelled monoclonal antibodies bound to specific antigens on the surface or in the cytoplasm of different cell populations of the immune system. Techniques from molecular biology and from cytogenetics are also relevant for the diagnosis of hematological diseases: they allow the identification of changes of the genetic material on the level of DNA (molecular biology) and chromosomes (cytogenetics). Molecular biological and cytogenetic methods coalesce in the field of molecular cytogenetics, which renders possible the identification of chromosome mutations, which are invisible by the classical cytogenetical approach, and difficult to detect by routine molecular biological analysis. Most hematological malignancies are associated with genomic changes, which can be identified by cytogenetic and/or molecular biological methods. These genetic changes usually correspond with a specific pattern of surface antigens of the tumour cells, which can be identified by FACS. The different mutations in different genes causing a large number of inherited hematological diseases can often be found by molecular analysis, too. For hematological neoplasias, the exact definition of the causative mutations is increasingly important for therapeutic decisions and follow-up analysis of minimal residual disease. For inherited diseases, the identification of mutations is often the basis for a correct genetic counselling of the family.

[Monoclonal gammopathies--from MGUS to myeloma]. / Monoklonale Gammopathien--vom MGUS zum Myelom.

Monoclonal gammopathies are characterized by the overproduction of a monoclonal immunoglobulin (M-Protein), which may be detected in serum or urine by protein electrophoresis and immunofixation. The presence of an M-Protein results from the proliferation of a single abnormal clone of differentiated B lymphocytes or plasma cells, and is associated with a variety of clinical conditions, ranging from asymptomatic to malignant disease. Recent years have witnessed considerable advances in the treatment of plasma cell myeloma, the most common malignant disorder of the monoclonal gammopathies. As compared with conventional-dose treatments, high-dose chemotherapy with autologous stem-cell transplantation increases response rates and overall survival of patients with myeloma who are younger than 65 years of age. Progress in supportive therapies and the development of promising new drugs such as proteasome inhibitors and thalidomide analogues may provide further benefits for myeloma patients in the future.

Therapeutic efficacy of FcgammaRI/CD64-directed bispecific antibodies in B-cell lymphoma.

CD64 (FcgammaRI) receptors represent highly potent trigger molecules for activated polymorphonuclear cells (PMN) and mediate lysis of a range of tumors in the presence of appropriate monoclonal antibodies. An huCD64 transgenic mouse model designed to analyze the therapeutic activity of a panel of bispecific F(ab')(2) (BsAb) in retargeting granulocyte-colony-stimulating factor (G-CSF)-activated PMN against syngeneic B-cell lymphomas is reported. This model allows careful analysis of the individual elements of the therapeutic process. BsAb were directed against immunoglobulin-idiotype (Id), major histocompatibility class II (MHC II), or CD19 on the tumors and huCD64 on the effectors. In vitro cytotoxicity assays and in vivo tumor tracking showed that, provided effectors were activated with G-CSF, all 3 derivatives destroyed and cleared lymphoma cells, with (huCD64 x MHC II) proving by far the most cytotoxic in vitro. However, though all derivatives delivered some survival advantage, only the [huCD64 x Id] BsAb provided long-term protection to tumor-bearing animals. These results demonstrate that CD64-recruited cytotoxic effectors operate in vivo but that the (huCD64 x Id) conferred an additional anti-tumor function essential for long-term protection. T-cell depletion studies demonstrated that this extra therapeutic activity with [huCD64 x Id] was totally dependent on CD4 and CD8 T cells and that mice, once "cured" with BsAb, were resistant to tumor rechallenge. These findings indicate that CD64 is an effective trigger molecule for delivering cytokine-activated PMN against tumor in vivo and that, provided tumor targets are selected appropriately, CD64-based BsAb can establish long-term T-cell immunity.

Antitumor immune effector mechanisms recruited by phage display-derived fully human IgG1 and IgA1 monoclonal antibodies.

We have constructed a recombinant, fully human IgA1 monoclonal antibody, UBS-54/IgA1, against the tumor-associated Ep-CAM molecule and compared its tumor-killing capacity with its IgG1 counterpart in in vitro assays. The data show that phage display-derived fully human IgA1 antibodies efficiently recruit immune effector cells that express the Fc receptor for IgA, FcalphaRI (CD89). UBS-54/IgA1-mediated killing of tumor cells by isolated polymorphonuclear cells (PMNs) and in whole blood was found to proceed without the necessity to preactivate effector cells with cytokines. In addition, the IgA1 anti-Ep-CAM human monoclonal antibody (huMab) triggered phagocytosis of tumor cells by monocyte-derived macrophages. Strikingly, simultaneous addition of IgA1 and IgG1 anti-Ep-CAM antibodies did not result in enhancement of tumor cell killing unless the effector cells were stimulated with granulocyte colony-stimulating factor. The lack of an additive effect could be attributed to an inhibitory effect of IgG on IgA-mediated tumor cell killing through binding of IgG1 to the inhibitory FcgammaRIIb receptor expressed by PMNs. These results show that IgA1 antitumor huMabs are capable of recruiting the large population of peripheral blood PMNs for tumor cell killing. This population is not effectively recruited by IgG type antibodies, currently the antibodies most frequently used for clinical application. In addition, the data suggest that a combination of IgG1 and IgA1 antitumor huMabs may collaborate in tumor cell killing in patients treated with granulocyte colony-stimulating factor.

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments.

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.

Murine IgG1 complexes trigger immune effector functions predominantly via Fc gamma RIII (CD16).

Previously, we have demonstrated that phagocytosis of IgG1-coated particles by macrophages in vitro is impaired by deletion of Fc gamma RIII in mice, suggesting that IgG1 may interact preferentially with Fc gamma RIII. In the present study, the biologic relevance of this observation was addressed by triggering various effector functions of the immune system in Fc gamma RIII(-/-) mice, using panels of mAbs of different IgG subclasses. Both binding and phagocytosis of IgG1-coated sheep or human erythrocytes by Fc gamma RIII(-/-) macrophages in vitro were strongly impaired, indicating that the impaired ingestion of complexed IgG1 by Fc gamma RIII(-/-) macrophages is due to a defect in binding. An in vivo consequence of the defective phagocytosis was observed by resistance of Fc gamma RIII-deficient mice to experimental autoimmune hemolytic anemia, as shown by a lack of IgG1-mediated erythrophagocytosis in vivo by liver macrophages. Furthermore, trapping of soluble IgG1-containing immune complexes by follicular dendritic cells in mesenteric lymph nodes from Fc gamma RIII(-/-) mice was abolished. Whole blood from Fc gamma RIII(-/-) mice was unable to induce lysis of tumor cells in the presence of IgG1 antitumor Abs. Finally, IgG1 mAbs proved unable to mount a passive cutaneous anaphylaxis in Fc gamma RIII(-/-) mice. Together, these results demonstrate that IgG1 complexes, either in particulate or in soluble form, trigger in vitro and in vivo immune effector functions in mice predominantly via Fc gamma RIII.

Mice lacking IL-12 develop polarized Th1 cells during viral infection.

Studies in IL-12-deficient mice established the necessity for IL-12 to generate a Th1 cytokine response that is often required for elimination of intracellular pathogens. In this study, we demonstrate that mice with a targeted disruption of the IL-12p40 and/or p35 gene effectively control liver damage induced by mouse hepatitis virus (MHV) infection, similar to wild-type animals. In contrast, MHV-infected IFN-gamma receptor-deficient (IFN-gammaR[-/-]) mice showed an increased susceptibility to coronaviral hepatitis. Surprisingly, MHV-infected mice lacking IL-12 produced a polarized Th1-type cytokine response, as evidenced by high IFN-gamma and nondetectable IL-4 production by CD4+ splenocytes and normal virus-specific serum IgG2a/IgG1 ratios. The virus-induced type 1 cytokine secretion pattern was not reversed in IL-12-deficient mice by in vivo neutralization of IFN-gamma nor in IFN-gammaR(-/-) mice receiving IL-12-neutralizing Abs. In IL-12-deficient mice, Th1-type responses were also generated upon immunization with inactivated MHV. In contrast, following immunization with keyhole limpet hemocyanin, mice lacking IL-12 mounted strongly reduced specific IgG2a and increased IgE responses, indicative of a type 2-dominated cytokine pattern. These findings demonstrate that following a virus infection, IL-12 is not essential for the generation of polarized T cell type 1 cytokine expression and associated immune responses, which is in marked contrast to nonviral systems. Our data suggest that viruses may selectively induce IFN-gamma production and Th1-type immune reactions even in the absence of IL-12.

Preclinical studies with Fc(gamma)R bispecific antibodies and granulocyte colony-stimulating factor-primed neutrophils as effector cells against HER-2/neu overexpressing breast cancer.

Immunotherapies directed to the proto-oncogene product HER-2/neu, which is overexpressed on a subset of breast and other carcinomas, currently receive considerable attention. We have investigated cell-mediated effector mechanisms of HER-2/neu antibodies against breast cancer cell lines. Compared to unfractionated control blood, whole blood from patients during granulocyte colony-stimulating factor (G-CSF) treatment exhibits significantly enhanced lysis (P < 0.001) of SK-BR-3 cells in the presence of HER-2/neu antibody 520C9. The extent of tumor cell killing correlated positively (r = 0.74) to polymorphonuclear neutrophil (PMN) blood counts. Fractionation of whole blood into plasma, mononuclear cells, and PMNs showed major killing capacity to reside in the granulocyte fraction. PMNs were efficiently cytolytic with a panel of HER-2/neu antibodies and against various breast cancer cell lines. Experiments with blocking antibodies to Fc(gamma)R documented Fc(gamma)RII (CD32) as the major trigger molecule for monoclonal antibody 502C9-mediated cytotoxicity. Killing via 520C9 was significantly influenced by an allotypic polymorphism of Fc(gamma)RIIa, the CD32 molecule expressed on PMNs. In reverse antibody-dependent cell-mediated cytotoxicity experiments with a panel of HER-2/neu-directed bispecific antibodies, Fc(gamma)RIII (CD16) proved to be an efficient trigger molecule in blood from healthy volunteers. During G-CSF treatment, however, Fc(gamma)RI (CD64)-expressed on monocytes and G-CSF primed, but not on healthy donor PMNs-became the predominant cytotoxic trigger molecule. Thus, G-CSF application increased effector cell numbers for HER-2/neu-directed immunotherapy, and G-CSF primed PMNs proved particularly effective with a [HER-2/neu x Fc(gamma)RI] bispecific antibody. These findings support clinical trials with HER-2/neu-directed antibodies in combination with G-CSF in breast cancer patients overexpressing HER-2/neu.

Human IgG Fc receptors.

Human IgG receptors constitute a family of glycoprotein complexes consisting of ligand-binding, and associated signaling chains. Three leukocyte classes (Fc gamma RI, II, and III) and one separate endothelial Fc gamma R class (FcRB) are defined which are expressed on hematopoietic and endothelial cells. Upon interaction with IgG, Fc gamma R initiate a plethora of signaling cascades involving receptor signaling motifs, and protein tyrosine kinases and phosphatases. These cascades ultimately culminate in activation or deactivation of effector cells, resulting in initiation or down-modulation of cellular processes. Recent evidence points to a crucial in vivo role of Fc gamma R in both initiation and regulation of inflammatory and cytotoxic responses. These Fc gamma R-mediated immune responses can be exploited to develop novel immunotherapies.

Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcgamma receptor I bispecific antibodies.

Abs are able to induce inflammatory antitumor responses by recruiting IgG Fc receptor (FcgammaR)-bearing cytotoxic effector cells. We recently described the capacity of the high affinity FcgammaRI (CD64) to trigger cytotoxic activity of neutrophils (PMN) during granulocyte CSF (G-CSF) treatment. To take advantage of FcgammaRI as a cytotoxic trigger molecule on PMN, two Ab constructs were prepared. We show that a chimeric human IgG1 Ab (Ch520C9) and an anti-FcgammaRI bispecific Ab (BsAb; 22x520C9), both directed to the proto-oncogene product HER-2/neu, interact with FcgammaRI. In addition, both Ab constructs mediate enhanced lysis of HER-2/neu-expressing tumor cells by G-CSF-primed PMN. However, engagement of FcgammaRI by Ch520C9 was inhibited by human serum IgG, thereby abrogating the enhanced Ch520C9-mediated cytotoxicity. BsAb 22x520C9, which binds FcgammaRI outside the ligand binding domain, effectively recruits the cytotoxic potential of FcgammaRI on G-CSF-primed PMN regardless of the presence of human serum. These results indicate that under physiologic conditions, serum IgG impairs activation of FcgammaRI-mediated cytotoxicity by conventional antitumor Abs. The IgG blockade can be circumvented with anti-FcgammaRI BsAbs. Using human FcgammaRI transgenic mice we demonstrate that BsAb 22x520C9 is able to engage FcgammaRI in vivo. BsAb 22x520C9 injected i.v. was readily detected on circulating PMN of G-CSF-treated transgenic animals. In addition, we showed that PMN remain "armed" with BsAb 22x520C9 during migration to inflammatory sites, and that after isolation such PMN specifically lyse HER-2/neu-expressing tumor cells. These results point to the possibility of targeting anti-FcgammaRI BsAbs to G-CSF-primed PMN in vivo, endowing them with specific anti-tumor activity.

Lysis of murine B lymphoma cells by transgenic phagocytes via a human Fc gamma RI x murine MHC class II bispecific antibody.

The class I IgG receptor (Fc gamma RI) on cytotoxic effector cells has been reported to initiate destruction of tumour cells by effector cells in vitro. We are aiming at developing an immunocompetent model to evaluate the cytotoxic capacity of human Fc gamma RI for the rejection of tumour cells in vivo. Therefore, we recently generated a transgenic mouse strain expressing human Fc gamma RI on monocytes, macrophages, and neutrophils. In these mice, the human receptor is up-regulated by granulocyte-colony-stimulating factor (G-CSF) and is able to trigger cellular responses. Subsequently, in the present study the B cell lymphoma IIA1.6 cell line is selected as a tumour target, and a human Fc gamma RI-directed antitumour bispecific antibody (bsAb) is constructed and characterized. Fab' fragments of mAb 22, which bind hFc gamma RI at an epitope that is distinct from the ligand binding site, were chemically linked to Fab' fragments of rat anti-(mMHC class II antigens) mAb M5/114, yielding bsAb 22 x M5/114. This bsAb was able to bind simultaneously to hFc gamma RI and mMHC class II antigens in a dose-dependent fashion. Binding of 22 x M5/114 to Fc gamma RI was not inhibited in the presence of human IgG. It is important to note that, MHC-class-II-expressing IIA1.6 lymphoma cells were lysed by whole blood from G-CSF-treated transgenic mice in the presence of bsAb 22 x M5/114. No lysis by whole blood from non-transgenic mice or from transgenic animals that had not received G-CSF was observed. These results indicate that human Fc gamma RI is able to mediate lysis of murine IIA1.6 lymphoma cells by transgenic effector cells via bsAb 22 x M5/114. A trial with transgenic mice, evaluating the efficacy of these hFc gamma RI-directed bsAb in combination with G-CSF for treatment of IIA1.6 B cell lymphoma, is currently in progress.

Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice.

The family of receptors for IgG (Fc gamma R) plays an essential role in antibody-mediated effector functions of the immune system. However, the specific contribution of each of the Fc gamma R classes to in vivo immune reactions is still unclear. Here, we demonstrate that mice deficient for the ligand-binding alpha chain of Fc gamma RIII lack NK cell-mediated antibody-dependent cytotoxicity and phagocytosis of IgG1-coated particles by macrophages. Strikingly, these mice lack IgG-mediated mast cell degranulation, are resistant to IgG-dependent passive cutaneous anaphylaxis, and exhibit an impaired Arthus reaction. These results indicate a prominent role for Fc gamma RIII in inflammatory and anaphylactic responses, making this receptor a potential target in immunotherapy.

FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo.

Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface-expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA-IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.