A multi-contrast MRI study of microstructural brain damage in patients with mild cognitive impairment.

OBJECTIVES: The aim of this study was to investigate pathological mechanisms underlying brain tissue alterations in mild cognitive impairment (MCI) using multi-contrast 3 T magnetic resonance imaging (MRI). METHODS: Forty-two MCI patients and 77 healthy controls (HC) underwent T1/T2* relaxometry as well as Magnetization Transfer (MT) MRI. Between-groups comparisons in MRI metrics were performed using permutation-based tests. Using MRI data, a generalized linear model (GLM) was computed to predict clinical performance and a support-vector machine (SVM) classification was used to classify MCI and HC subjects. RESULTS: Multi-parametric MRI data showed microstructural brain alterations in MCI patients vs HC that might be interpreted as: (i) a broad loss of myelin/cellular proteins and tissue microstructure in the hippocampus (p ≤ 0.01) and global white matter (p < 0.05); and (ii) iron accumulation in the pallidus nucleus (p ≤ 0.05). MRI metrics accurately predicted memory and executive performances in patients (p ≤ 0.005). SVM classification reached an accuracy of 75% to separate MCI and HC, and performed best using both volumes and T1/T2*/MT metrics. CONCLUSION: Multi-contrast MRI appears to be a promising approach to infer pathophysiological mechanisms leading to brain tissue alterations in MCI. Likewise, parametric MRI data provide powerful correlates of cognitive deficits and improve automatic disease classification based on morphometric features.

Weekly x 4 induction therapy with the anti-CD20 antibody rituximab: effect on circulating t(14;18)(+) follicular lymphoma cells.

Rituximab 375 mg/m(2) weekly x 4 has been reported to induce a 60% response rate in patients with relapsed follicular lymphomas (FL). Our aim was to examine the effect of this rituximab schedule on circulating FL cells in an ongoing multicenter study. One hundred fifty-four patients with FL were examined by nested polymerase chain reaction (PCR) at baseline for the presence of t(14;18) translocation-carrying lymphoma cells in bone marrow and/or blood. Sixty-four patients (42%) had PCR-detectable t(14;18)(+) FL cells. Pretreatment characteristics of these 64 patients were as follows: one had stage I, nine had stage II, 14 had stage III, and 40 had stage IV disease. Thirty-five patients had bulky disease (> or = 5 cm) and 25 patients had an elevated serum lactate dehydrogenase (LDH) level. Bone marrow was morphologically assessed in 64 patients, and 39 of these patients had an infiltration with FL cells. Blood samples from 51 patients were available for PCR analysis between weeks 8-12 after induction therapy, and 28 of these patients (55%) were PCR negative. Paired blood and bone marrow samples were available for PCR analysis from 39 patients between weeks 8-12 after induction therapy with rituximab. Thirteen of these patients (33%) did not have PCR-detectable cells in blood and bone marrow, while 26 patients (67%) still had circulating t(14;18)(+) cells in either bone marrow (eight patients), blood (one patient), or both (17 patients). PCR negativity in blood and bone marrow in 13 patients was statistically significantly associated with partial or complete response after induction therapy with rituximab (P = 0.006). However, clearance of PCR-detectable t(14;18)(+) cells in bone marrow and/or blood could not be associated with any low tumor burden pretreatment characteristics such as stages I/II, absence of morphological bone marrow infiltration or tumor bulk of > or = 5 cm, and normal serum LDH.

The effect of Rituximab on patients with follicular and mantle-cell lymphoma. Swiss Group for Clinical Cancer Research (SAKK).

BACKGROUND: Clinical activity of the anti CD-20 monoclonal antibody Rituximab has been reported in patients with follicular lymphoma (FL) and mantle-cell lymphoma (MCL). PATIENTS AND METHODS: 120 patients with bi-dimensionally measurable FL or MCL (R.E.A.L. Classification) were treated with Rituximab 375 mg/m2/week for 4 weeks. A central pathology review confirmed the diagnosis of FL in 76 of 78 and of MCL in 39 of 42 cases. The response was evaluated after 8 weeks and confirmed after 12 weeks from the start of treatment. RESULTS: The toxicity of the treatment was, as expected, grade 1-2 fever and rigors during the first infusion and mild asthenia during the treatment period. Serious adverse events, probably or possibly related to the study treatment, included four deaths (3 of cardiac origin, 1 caused by P. carinii pneumonia) and 10 further nonfatal cases, including a permanent agranulocytosis and one case of heart failure. Response rate at week 12 was 52% for FL and 22% for MCL. After treatment, the BCL-2 rearrangement disappeared in 15 of 29 blood but only in 5 of 23 bone marrow samples; BCL-1 disappeared in 5 of 12 blood and 0 of 7 bone marrow specimens, as determined by PCR. CONCLUSIONS: Rituximab is an active agent for the treatment of FL, while its efficacy is modest in MCL. The effect in reducing minimal residual disease is more pronounced on the blood than it is on the bone marrow.

Involvement of the CD27-CD70 co-stimulatory pathway in allogeneic T-cell response to follicular lymphoma cells.

Non-activated follicular lymphoma (FL) cells do not function as effective antigen-presenting cells in vivo. CD40 activation of FL cells up-regulates critical adhesion and co-stimulatory molecules, thereby inducing lymphoma-specific cytotoxic T cells in vitro. However, other evidence suggests that CD70 is another important co-stimulatory molecule involved in antigen dependent T-cell activation. Here, we showed that freshly isolated FL cells from eight diagnostic biopsies expressed intermediate to high levels of CD27, whereas only insignificant levels of CD70 were detected on their cell surface. Together with the low to intermediate expression of B7, these findings help to explain the poor antigen-presenting capacity of non-activated FL cells. Activation of FL cells by CD27 and CD40 induced a significantly higher alloantigen T-cell response than CD40 alone, whereas CD27 activation induced only a mostly insignificant T-cell proliferation. Both CD40 and CD27 + CD40 activation resulted in a high up-regulation of CD70 and B7 molecules, whereas CD27 activation up-regulated CD70 but not B7 expression on the surface of FL cells. Thus, expression of both CD70 and B7 co-stimulatory molecules appears to be essential for an efficient T-cell-mediated immune response. However, the molecules responsible for the significantly higher alloantigen T-cell response to FL cells activated through CD27 and CD40 remain to be identified. In summary, we conclude that CD27+CD70 represents another co-stimulatory pathway involved in T-cell-mediated immune responses to FL cells. Our findings suggest that several co-stimulatory pathways exist and should be taken into consideration to optimize antigen presentation for the generation of lymphoma-directed cytotoxic T cells for adoptive immunotherapy.

Selection and immunomagnetic purging of peripheral blood CD34+ cells for autologous transplantation in B-cell non-Hodgkin's lymphomas.

BACKGROUND: Clonogenic tumor cells in the hematopoietic progenitor cell harvest may contribute to relapse after high dose therapy for B-cell malignancies. Purging of the HPC harvest requires large amounts of anti-B-cell antibodies, whereas CD34-selection enriches self renewing HPC's but malignant cells are still detectable in many CD34+ fractions. PATIENTS AND METHODS: We examined the feasibility and safety of a CD34-selection followed by purging with anti B-cell antibodies in 11 patients with B-cell non-Hodgkin's lymphomas undergoing high-dose therapy with cyclophosphamide, BCNU and etoposide with retransfusion of autologous HPC's. RESULTS: A mean number of 340 x 10(8) mononuclear cells was used for CD34-selection and immunomagnetic purging. CD34+ cells were enriched from a mean of 1.7% (range 0.2%-4.5%) to a mean of 68% (range 49%-87%) with a mean recovery of 27% (range 15%-43%). The mean number of retransfused CD34+ cells was 1.2 x 10(6)/kg (range 0.6-2.2 x 10(6)/kg) body weight with a median of 11 days (range 10-13 days) to neutrophil recovery of 0.5 x 10(9)/l and 17 days (range 13-25 days) to platelet recovery of 50 x 10(9)/l. Mean number of intravenous antibodies and inpatient days were 8 (range 0-14) and 22 (range 19-26) respectively. Major toxicity consisted in four septicemias. CONCLUSIONS: CD34-selected and purged HPC's are safe and mediate rapid hematological recovery after high dose therapy for B-cell non-Hodgkin's lymphomas.

T-cell derived cytokines co-stimulate proliferation of CD40-activated germinal centre as well as follicular lymphoma cells.

Follicular lymphomas, the malignant counterparts of normal germinal centre (GC) B-cells, grow in vivo in close association with polyclonal T-cells, predominantly from the T-helper cell type. T-cell-derived growth factors are involved in the development of GC B-cells. However, their role in the pathogenesis of follicular lymphomas has not been clearly defined. We investigated the co-stimulatory activity of 14 cytokines (interleukin-1 to -8, IL-10, IL-13, INF-alpha, TNF-alpha, GM-CSF and SCF) on the proliferation of CD40-activated follicular lymphoma cells in comparison to tonsillar GC B-cells. Tonsillar GC B-cells (n = 4), malignant cells from diagnostic lymph node biopsies of patients with follicular (n = 4) or transformed (n = 4) lymphomas were grown on irradiated CD40-ligand transfectants, with and without cytokines. [3H]-thymidine uptake was measured at day 7. IL-10 and IL-4 proved to be the most potent co-stimulators of proliferation of tonsillar GC B-cells, whereas proliferation of follicular lymphoma cells was co-stimulated by IL-4. The fact that IL-4 is a T-cell derived cytokine, suggests that lymphoma infiltrating T-cells play a role in the growth of these malignancies. Moreover, proliferation of both non-neoplastic tonsillar GC B-cells and follicular lymphomas is co-stimulated by T-cell derived cytokines, indicating that responsiveness to paracrine factors may not be a characteristic of the malignant phenotype.

Lung tumor cells: a multivariate approach to cell classification using two-dimensional protein pattern.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful research tool for the analytical separation of cellular proteins. The qualitative and quantitative pattern of polypeptides synthesized by a cell represents its phenotype and thus defines characteristics such as the morphology and the biological behavior of the cell. By analyzing and comparing the protein patterns of different cells it is possible to recognize the cell type and also to identify the most typical features of these cells. In applied pathology it is often difficult to identify the tissue of origin and the stage or grade of a neoplasia by cellular morphology analyzed by classical or immunostaining procedures. The protein pattern itself is the most characteristic feature of a cell and should therefore contribute to the identification of the cell type. For this reason we separated protein fractions originating from different lung tumor cell lines using 2-D PAGE and we compared the resulting patterns on a multivariate statistical level using correspondence analysis (CA) and ascendant hierarchical clustering (AHC). The results indicate that (i) protein patterns are highly typical for cells and that (ii) the comparison of the protein patterns of a set of interesting cell types allows the identification of potentially new marker proteins. 2-D PAGE is thus a unique and powerful tool for molecular cytology or histopathology, unveiling the protein expression level of tissues or cells.

Radiation studies on B cell differentiation marker CD24/SCLC Cluster-4 antigen expressing and non-expressing lung cancer cell lines and mouse fibroblasts.

A correlation between CD24 expression and higher intrinsic radiation sensitivity has been described in B-lineage acute lymphoblastic leukaemia (B-ALL). We recently identified the SCLC surface antigen Cluster-4 (CL-4) to be identical to the B cell differentiation marker CD24, except for one amino acid residue. The CD24/CL-4 antigen is highly expressed on SCLC, but rarely on NSCLC cells. In order to investigate the influence of the expression of CD24/CL-4 on the radiation sensitivity in a non-leukaemic cell system, sublines of the human SCLC H249 cell line transfected with mutated ras oncogene, and differing in their CD24/CL-4 expression, were studied. In addition, we stably transfected the NSCLC A125 cell line and the mouse fibroblast NIH3T3 cell line with the CL-4 cDNA. The differential expression of CD24/CL-4 on the cells had no influence on morphology, proliferation and cloning efficiency. Radiation studies were done with cells in exponential growth phase. In the highly resistant NSCLC A125 cells no difference in radioresponsiveness was observed between CD24/CL-4 expressing and non-expressing cells. In the rather radiosensitive cells, similar responses to radiation were observed between CD24/CL-4 expressing and non-expressing SCLC H249-ras cells, whereas the CL-4 transfected NIH3T3 mouse fibroblasts showed a substantially higher radioresistance than the CD24/CL-4 non-expressing control cells. In conclusion, the correlation between CD24/CL-4 expression and radiation sensitivity is controversial and depends on the cell type.

Autocrine stimulation of a human lung mesothelioma cell line is mediated through the transforming growth factor alpha/epidermal growth factor receptor mitogenic pathway.

Malignant cells frequently acquire a certain independency of exogenous growth factors via the coexpression of epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF)-related molecules. In the present study we investigate a possible involvement of EGF-related molecules in the growth of human lung mesothelioma. Four well-characterised cell lines are analysed for their responsiveness to exogenous EGF and transforming growth factor alpha (TGF-alpha) as well as for coexpression of EGFR and EGF/TGF-alpha. Both growth factors are able to stimulate DNA synthesis in three cell lines, although the degree of responsiveness is very variable, but neither EGF nor TGF-alpha has an effect on the cell line ZL34. In contrast, no heterogeneity is observed in the expression of EGFR, which is similarly high in all cell lines. Analysis of cell supernatants reveals that, whereas no EGF is detected, TGF-alpha is released by two cell lines. Furthermore, these two cell lines, ZL5 and ZL34, are shown to express the membrane anchored precursor pro-TGF-alpha. Thus, coexpression of EGFR and TGF-alpha is observed on two mesothelioma cell lines. The potential autocrine mitogenic role of TGF-alpha in these two cell lines was tested using neutralising antibodies against TGF-alpha and EGFR. In ZL5 cells DNA synthesis was not affected by the presence of neutralising antibodies, indicating that an external autocrine mitogenic pathway is not active in these cells. In ZL34 cells, however, the potential autocrine loop could be disrupted, as DNA synthesis was significantly reduced in the presence of neutralising antibodies. This result gives strong evidence for an autocrine role of TGF-alpha in the growth of the mesothelioma cell line ZL34.

In vitro effect of suramin on lung tumour cells.

In the search for new therapeutic concepts in lung cancer chemotherapy, suramin, a potential anticancer drug which evades multidrug resistance, was tested in vitro on 25 lung-derived cell lines, either non-tumorigenic cells, or established cell lines from five different tumour types. Suramin treatment resulted in a time- and dose-dependent decrease in [3H]thymidine incorporation, except in one adenocarcinoma cell line where DNA synthesis was highly stimulated. [3H]Leucine incorporation was less affected, indicating that suramin acted cytostatically rather than cytotoxically. Our results show that suramin affected DNA synthesis of the different types of lung derived cells, including non-tumorigenic and tumour cell lines, to a similar extent.

Hematopoietic growth factors secreted by seven human pleural mesothelioma cell lines: interleukin-6 production as a common feature.

Seven mesothelioma cell lines, established from patients with pleural mesothelioma, exhibited substantial heterogeneity regarding in vitro morphology and growth characteristics. Media conditioned by these cell lines and by MeT5A normal mesothelial cells were examined for (i) colony formation on human bone-marrow cells, (ii) hematopoietic growth-factor content and (iii) mitogenic activity on mesothelioma cells. Colony-stimulating activity was produced only by the ZL34 cell line. Analysis of conditioned media by ELISA revealed that all mesothelioma cell lines constitutively produced IL-6, while the MeT5A normal mesothelial cells did not; in addition, GM-CSF and G-CSF were detected in the supernatant of the ZL34 cell line. Using a 3H-thymidine incorporation assay, we showed that all mesothelioma cell lines produced mitogenic activity in the culture supernatant, in contrast to the MeT5A normal mesothelial cells. The mitogenic effect of the hematopoietic growth factors detected in mesothelioma culture supernatants was tested on mesothelioma cells and on MeT5A normal mesothelial cells: IL-6, GM-CSF and G-CSF did not stimulate any DNA synthesis. Our results suggest that these hematopoietic growth factors do not act as autocrine growth factors. A common feature of this panel of mesothelioma cell lines is the production of IL-6; although the biological significance of the aberrant production of cytokines by mesotheliomas remains unclear, IL-6 might be involved in paraneoplastic syndromes such as thrombocytosis.

An autocrine mitogenic activity produced by a pleural human mesothelioma cell line.

The pleural human mesothelioma cell line ZL5 established in our laboratory exhibits an unusual phenotype with adherent and floating cells. ZL5 cells grow in a chemically defined medium (ACL3*) and can be maintained over 3 weeks in protein-free basal medium alone (RPMI). Basal medium conditioned by ZL5 cells possesses a mitogenic activity with an autocrine effect, as measured by cell counting and by a 3H-thymidine incorporation assay. Moreover, the conditioned medium affects the DNA synthesis of a variety of other lung-derived cells. The active principle of medium conditioned by ZL5 cells is not identical to the defined growth-factors EGF, PDGF, and TGF-beta, known to stimulate the growth of normal human mesothelial cells: treatment with these factors does not mimic the effect of conditioned medium on ZL5 cells. Our observations suggest that the mesothelioma cell line ZL5 produces an unknown autocrine mitogen.

Analysis of the structural diversity of monoclonal antibodies to cyclosporine.

The immunosuppressive cyclic undecapeptide cyclosporine (Cs) represents a useful model for studying the molecular basis of antibody-antigen interactions. The three-dimensional structure of the Cs molecule is known and a large panel of monoclonal antibodies (mAbs) to Cs has been well characterized by cross-reactivity studies with numerous Cs analogs. In the present study, the sequences of the variable regions of seven mAbs to Cs were determined and a striking relationship was found between the expressed variable region genes and the Cs recognition pattern. An analysis of the length and hydrophobic content of the hypervariable regions and sequence similarities suggested that the heavy chain plays a major role in Cs recognition. Different fine specificities were observed for mAbs exhibiting identical light chains, while two antibodies differed by only a single amino acid located in the heavy chain. The presence of a duplication of 12 nucleotides within the heavy chain third hypervariable region of two antibodies suggests the existence of an additional mechanism for creating antibody diversity.

Monoclonal antibodies to cyclosporin are representative of the major antibody populations present in antisera of immunized mice.

Two series of mouse antisera raised against cyclosporin (Cs)-carrier conjugates exposing opposite sides of the Cs molecule and more than sixty monoclonal antibodies (mAbs) derived from the same animals were compared in terms of isotype and fine specificity for Cs. The predominant isotypes of the mAbs reflected the in situ distribution of the circulating anti Cs antibodies. The fine specificity of the antibodies was studied by determining their cross-reactivity for a series of Cs-derivatives and Cs-metabolites in competitive ELISA. The antisera raised by different immunizations showed very different cross-reactivity patterns for the Cs-derivatives. However, the in situ anti Cs antibody populations and the majority of mAbs derived from the corresponding animals showed a striking similarity in fine specificity for restricted clusters of residues on the Cs molecule. These results indicate that the mAbs produced against Cs are representative of the major antibody population present in the sera of the mice used for the fusion. By determining the characteristics of antibodies found in the serum of immunized mice it may thus be possible to select animals that are likely to give rise to mAbs of a certain isotype and specificity.

Crystallization and preliminary X-ray investigation of a complex between a Fab fragment and its antigen, cyclosporin.

Preliminary crystallographic data are given for a complex between the cyclic undecapeptide cyclosporin and the Fab fragment of an anti-cyclosporin monoclonal antibody. Crystals of the complex are orthorhombic with space group P2(1)2(1)2(1) and diffract to 2.7 A resolution. The unit cell dimensions are a = 52.6 A, b = 70.2 A and c = 118.4 A. A native data set to 2.7 A resolution has been collected.

Study of the conformation of cyclosporine in aqueous medium by means of monoclonal antibodies.

The three-dimensional structure of the immunosuppressive cyclic peptide cyclosporine (Cs), determined in crystal by X-ray analysis and in solution in aprotic solvents by n.m.r., differs mainly by the orientation of the 7 carbon side chain of residue 1. Because of its poor solubility in water, the conformation of Cs in aqueous medium cannot be studied by n.m.r. methods, which require concentrations of the substance of the order of milligram/mL but can be analyzed by immunochemical methods in which concentrations in the nanogram/mL range are detected. In the present study, the ability of a series of monoclonal antibodies (McAbs) raised against Cs to recognize different parts of residue 1 of Cs was determined from the cross-reactivity of different Cs-analogues modified in residue 1. The results show that when Cs is dissolved in aqueous buffer, the terminal atoms of residue 1 side chain are not available for binding to antibodies recognizing the face of the molecule defined by residues 1, 2, 3, 10, 11, suggesting that the chain is probably folded back under the molecule, as observed in the crystal structure. Binding of McAbs to Cs was also affected by conformational modifications of the peptide ring that occur in some Cs-analogues. The results illustrate the potential of McAbs for probing the conformation of Cs-derivatives for which no structural data are available.