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Biomedical subjects

W H Fridman

Publications and source records attributed to W H Fridman.

At least 73 records · Page 4Linked to original sources
PubMed1998

Major histocompatibility complex class I presentation of exogenous soluble tumor antigen fused to the B-fragment of Shiga toxin.

Targeting exogenous antigen into the MHC class I-restricted presentation pathway is a prerequisite for the induction of cytotoxic T lymphocytes (CTL) which have been shown to represent an important component of the protective and therapeutic immune response to viral infections and tumors. In this study, we produced recombinant proteins composed of the receptor-binding non-toxic B-fragment of bacterial Shiga toxin derived from Shigella dysenteriae associated with an epitope from a model tumor antigen, Mage 1. We show that Shiga B-Mage 1 fusion proteins carrying an active or inactive endoplasmic reticulum retrieval signal (the C-terminal peptides KDEL or KDELGL, respectively) could be presented by peripheral blood mononuclear cells in an MHC class I-restricted manner to Mage 1-specific CTL. After pulsing B lymphoblastoid cells or dendritic cells with Shiga B-Mage 1 fusion protein, activation of the MHC class I-restricted Mage 1-specific CTL was also demonstrated. In further analysis, we showed that treatment with brefeldin A or paraformaldehyde fixation of Epstein-Barr virus-transformed B cells prevented the presentation of the Mage 1 T cell epitope, which excluded extracellular processing of the antigen. Immunofluorescence analysis also revealed that the Shiga B-Mage 1 fusion protein was largely excluded from Lamp-2-positive lysosomal structures. Therefore, the ability of Shiga toxin B-fragment to target dendritic cells and B cells and to direct antigen into the exogenous class I-restricted pathway makes it an attractive non-living and non-toxic vaccine vector.

Antigen Presentation↗
PubMed1998

Gene therapy with cytokine-transfected xenogeneic cells in metastatic tumors.

On the basis of compelling preclinical data in cats and dogs we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human IL-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to CT- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. Endpoints of the study were feasibility, toxicity, and clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological and molecular analyses were performed on biopsy specimens of tumors and blood samples from before, during and after treatment. Low levels of serum antibodies to Vero cells developed in 2/9 patients. Analysis of tumor biopsies showed increased expression of CD3 mRNA and enhanced tumor infiltration with varying lymphocyte subpopulations after treatment. In addition, monoclonal alterations of the TCR repertoire of blood and tumor lymphocytes were observed. Treatment was well tolerated and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft tissue sarcoma showed a more than 90% and more than 50% reduction of the volume of two distant, non-injected metastases, respectively, lasting for 22+ months. Four other patients showed stabilization of their disease for three to nine months, among whom was a patient with melanoma who developed marked vitiligo. We conclude that repeated injection of up to 5 x 10(7) Vero-IL-2 cells was safe and showed biological and clinical activity in heavily pretreated patients with advanced solid tumors. Further evaluation of intratumoral application of Vero-IL-2 seems warranted.

Adult↗
PubMed1998

Identification of two quantitative trait loci involved in antibody production on mouse chromosome 8.

Several quantitative trait loci (QTLs) contributing to the extreme phenotypes of the selected high (H) and low (L) antibody-responder lines of mice were mapped on distinct chromosomes. Successive backcrosses were bred to reduce the length of the QTL-bearing segment detected on chromosome 8 and to produce congenic lines to test gene effect independently of the other QTLs. An increase in antibody responses was repeatedly found to be associated with inheritance of the H-line allele at two markers separated by 30 cM on that chromosome. In the successive backcrosses, background and unlinked involved genes of H-line origin were progressively eliminated; however, unexpected within-progeny variations persisted in the third and even fourth backcross. Nevertheless, the presence of two QTLs within the considered interval was definitely demonstrated in distinct progenies of the fourth backcross which separately inherited one of the two gene-marker H-line alleles. The previously identified chromosome 8 segment therefore contains at least two QTLs involved in antibody responsiveness.

Animals↗
PubMed1998

Structurally different anthracyclines provoke different effects on cell cycle and tumor B cell differentiation.

Previously we have detected a stimulatory effect on immunoglobulin (IgG) synthesis when hybridoma cells were treated with doxorubicin. In order to determine whether this is a general property of anthracycline, we have selected three analogs--doxorubicin (DOX), pirarubicin (THP-DOX) and aclarubicin (ACR)--which differ mainly in the methylation state of their amino sugars. Cell cycle analysis by flow cytometry and drug localization by scanning confocal microscopy were also performed. The results show that when cells (UN2 hybridoma B cells), were exposed to subtoxic doses of DOX or THP (with unmethylated amino sugars), a strong increases in IgG secretion, heavy (H) and light (L) chain synthesis and the corresponding mRNA levels were induced. Furthermore these two drugs arrested the cells in the G2/M phase of the cell cycle. In contrast, exposure to ACR (with its methylated amino sugar) at similar subtoxic doses induced a blockade of cells in the G1 phase with no increase of IgG synthesis, at the subtoxic doses used, all three drugs could still be detected in the nucleus as well as in the cytoplasm, as determined by confocal laser microscopy. Thus, the relationship between cell cycle blockade, IgG stimulation and anthracycline structure is suggested by these results.

Aclarubicin↗
PubMed1998

Cytokines and cancer.

The relationships between cytokines and cancer are multiple and bidirectional. On the one hand, cytokines may directly influence carcinogenesis and metastasis by modifying the tumor phenotype. On the other hand, during tumor progression, modifications of the cytokine expression in the tumor environment may be induced by the tumor cells, leading to a state of immunosuppression reflected by low cytokine expression in tumor stroma. Cytokines also play a role by stimulating the host immune system to generate anti-tumor specific responses. Finally, the use of cytokines as anti-tumor agents has led to objective clinical responses in about 15-25% of patients with metastatic melanoma or renal cell carcinoma, which presents the basis for the development of promising immunotherapeutic approaches for cancer therapy.

Animals↗
PubMed1998

p53 status does not predict initial clinical response to bacillus Calmette-Guerin intravesical therapy in T1 bladder tumors.

PURPOSE: In superficial urothelial tumors of the bladder, p53 status is currently the most informative pretreatment parameter to define a population at higher risk for invasive carcinoma. Also, in T1 tumors, occurrence of muscular invasion is often related to an early relapse following BCG therapy. With the knowledge of biological parameters able to identify the group of initial BCG therapy non-responders, it would be possible to offer earlier treatment to the patients who need a more aggressive mode of therapy. The aim of this work was to study the predictive value of the p53 tumor status on the early BCG therapy response. MATERIALS AND METHODS: The population included a selected group of 43 patients presenting T1 bladder tumors with no carcinoma in situ (Tis), treated by transurethral resection (TUR) followed by intravesical BCG therapy. Clinical outcome was analyzed in relation to usual clinical and histopathological parameters, and pretreatment p53 tumor status was assayed by an immunohistochemical technique using DO7 monoclonal antibody. For 16 specimens, p53 gene was investigated using a Single Strand Conformation Polymorphism (SSCP) analysis and sequence determination. RESULTS: p53 anomalies were strongly correlated to smoking behavior (p = 0.003) and tumoral grade (p = 0.025). Univariate analysis revealed an absence of correlation between p53 immunostaining and initial, one and two years response-rate to BCG therapy. However, longterm followup revealed a trend between positive staining and disease progression. The p53 molecular study validated the use of DO7 immunostaining in detection of p53 anomalies. CONCLUSIONS: In T1 bladder tumors, pretreatment p53 determination was not useful to define a group of early BCG non-responders. Thus, p53 status and immunological response induced by BCG endovesical therapy are two independent events.

Adult↗
PubMed1998

[Fas, fas ligand, immune tolerance, and cancer: implications in cancer of the colon].

Fas and Fas ligand (FasL) are implicated in programmed cell death or apoptosis. In the immunological field, they are particularly important in auto-immunity, graft rejection and anti-tumoral response. Fas ligand expression on thymocytes, activated T lymphocytes, and in sites of immune privilege, suggests the importance of Fas/FasL interactions in negative control of the immune response. The recent description of FasL expression by tumoral cells, represents a new mechanism of immune escape for different cancer, and has been well studied in colon adenocarcinoma.

Apoptosis↗
PubMed1997

Effect of TGF-beta1 on cell cycle regulatory proteins in LPS-stimulated normal mouse B lymphocytes.

The cell cycle events accompanying TGF-beta1-induced growth arrest of normal mouse resting B lymphocytes stimulated by LPS were investigated. We showed that TGF-beta1 prevents the retinoblastoma protein (pRb) phosphorylation and induces growth arrest in mid- to late G1. To explore the molecular basis of the effect of TGF-beta1, we analyzed the in vitro kinase activities of cyclin/cyclin-dependent kinase (cdk) complexes involved in the progression through G1 phase and in the G1/S transition, by using the glutathione S-transferase-pRb fusion protein as a substrate. Cdk2-associated kinase activity was strongly induced in mitogen-treated B cells. It was dramatically inhibited by TGF-beta1 as were the cyclin E- and cyclin A-dependent kinase activities. TGF-beta1 treatment had no significant effect on the expression of two G1/S phase proteins, cyclin E and cdk2. In contrast, the appearance of cyclin A, occuring in late G1 phase, was almost totally inhibited by TGF-beta1. We also showed that expression of the cdk inhibitor protein p27Kip1 decreased as cells progressed through the G1 phase. An accumulation of p27 was found in TGF-beta1-treated cells, showing that TGF-beta1 prevented LPS-induced decline of p27. Finally we found that the lack of kinase activity associated with cyclin E/cdk2 complexes was correlated with increased amounts of cdk2- and cyclin E-bound p27. Overall, these results suggest that both cyclin A and cdk2 may be active participants in the TGF-beta1-induced cell cycle arrest in normal mouse B cells and indicate the involvement of p27 in this mechanism.

Animals↗
PubMed1997

Soluble interleukin-2 receptor serum level as a predictor of locoregional control and survival for patients with head and neck carcinoma: results of a multivariate prospective study.

BACKGROUND: The diagnosis and follow-up of head and neck carcinoma patients are based exclusively on clinical staging, which cannot always predict clinical outcome accurately. Because oral squamous cell carcinomas produce interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha and express IL-2 receptors, the authors assessed the prognostic value of the serum levels of these markers. METHODS: Serum levels of IL-6, TNF-alpha, soluble IL-2 receptors (s-IL-2-R), and acute phase proteins were measured at the time of diagnosis in a prospective study of 85 patients with primary squamous cell carcinoma of the head and neck. The influence of each clinical and laboratory parameter on locoregional control and survival was analyzed. RESULTS: At presentation, a relationship was observed between advanced tumor (T) classification and high serum levels of CRP (P = 0.0015) and s-IL-2-R (P < 0.05). A high lymph node (N) classification was significantly associated with elevated serum IL-6 (P = 0.01) and CRP levels (P = 0.0002). In the univariate analysis, T classification, N classification, performance status, Prognostic Inflammatory and Nutritional Index, and serum s-IL-2-R level were significantly correlated with both locoregional control and survival. Multivariate analysis showed that the only significant prognostic factors related independently to locoregional control were N classification (P = 0.02) and serum s-IL-2-R level (P = 0.02). In a Cox multivariate analysis, serum s-IL-2-R level was found to be the most predictive factor of survival (P = 0.0001). CONCLUSIONS: This study shows that serum s-IL-2-R level at the time of diagnosis represents a new independent prognostic variable for predicting the risk of locoregional recurrence and survival for patients with head and neck squamous cell carcinoma.

Adult↗
PubMed1997

Affinity of the interaction between Fc gamma receptor type III (Fc gammaRIII) and monomeric human IgG subclasses. Role of Fc gammaRIII glycosylation.

Binding of the Fc region of IgG antibodies to low affinity Fc gamma receptors (Fc gammaR) triggers important effector functions in the immune system. The type IIIb Fc gammaR (Fc gammaRIIIb or CD16) is a heavily glycosylated protein anchored to the membrane of neutrophils by a glycosylphosphatidylinositol link. This receptor contributes to cell activation by IgG immune complexes. To better understand the nature of the ligand-receptor association, we have studied the affinity and kinetics of the interaction between human IgG subclasses and two soluble forms of Fc gammaRIIIb (sFc gammaRIIIb or sCD16) corresponding to the 188 N-terminal amino acids of the extracellular region of the receptor, a glycosylated one made in eucaryotic cells (euc.sCD16) and a non-glycosylated one (proc.sCD16) made in Escherichia coli. Experiments using a BIAcore instrument, to measure protein binding in real time, showed that monomeric human IgG1 and IgG3, but not IgG2, IgG4, IgA and divalent antigen-binding fragments (F(ab')2) of IgG1, bound to immobilized euc.sCD16 with an affinity constant (K(A)) of 1.3 +/- 0.6 x 10(6) M(-1) and 2.6 +/- 0.4 x 10(5) M(-1), respectively. The affinity constant of proc.sCD16 for human IgG1 was in the same range (1.1 +/- 0.2 x 10(6) M(-1)), whereas that for human IgG3 was twofold higher (4.2 +/- 0.4 x 10(5) M(-1)). The specificity of the non-glycosylated receptor for human IgG subclasses bound to Sepharose was IgG1 > IgG3 >> IgG4 >>> IgG2. Thus, the extracellular polypeptide of Fc gammaRIIIb dictates the interaction of the receptor with IgG subclasses although glycosylation plays an inhibitory role in the interaction with human IgG3.

Animals↗
PubMed1997

Soluble CD16/Fc gamma RIII induces maturation of dendritic cells and production of several cytokines including IL-12.

Fc gamma RIII (CD16), a low affinity FcR which binds IgG-containing immune-complexes, exists under membrane-associated forms and under a soluble form (sFc gamma RIII). The latter, present in biological fluids (serum, saliva), is generated by proteolytic cleavage of the two membrane-associated Fc gamma RIII isoforms, Fc gamma RIII-A (expressed by macrophages and NK cells) and Fc gamma RIII-B (expressed exclusively by neutrophils). Herein we demonstrate that dendritic cells (DCs), generated by culturing monocytes with GM-CSF and IL-4, bind biotinylated recombinant sFc gamma RIII. This binding is specific and involves the complement receptor CR3 (CD11b/CD18) and CR4 (CD11c/CD18). Indeed, preincubation of DCs with anti-CD11b and anti-CD11c mAbs decreased by 52% and 62% respectively the binding with sFc gamma RIII. Moreover, electron microscopy showed that binding of gold-labeled sFc gamma RIII to DCs maintained at 4 degrees C occurred within clathrin-coated pits. Once internalized, at 37 degrees C, sFc gamma RIII entered the endocytic pathway and reached the MHC class II compartments. Furthermore, DCs incubated for 48 h with multivalent sFc gamma RIII expressed increased levels of CD40, CD80, CD86, CD54, CD58, HLA class I and class II molecules and decreased levels of CD23 and CD32. These effects result in an increased capacity of DCs to trigger proliferative responses by CD4+ CD45RA+ allogeneic T cells. RT-PCR amplification demonstrated that incubation of DCs for 20 h in the presence of multivalent sFc gamma RIII induced the appearance of GM-CSF and IL-12 p40 mRNA. Among the cytokines constitutively expressed, IL-1 beta and IL-8 were strongly up-regulated whereas IL-6 and IL-12 p35 mRNA were increased to a lesser extent and the expression of MIP-1 alpha mRNA remained constant. Finally, ELISA tests demonstrated that DCs incubated with multivalent sFc gamma RIII secreted the cytokines IL-1 beta, IL-6, IL-8, GM-CSF and IL-12 p75. Thus, while becoming internalized sFc gamma RIII could affect the capacity of DCs to present antigens and, via the induction of accessory molecules and the release of the IL-12 p75 protein, could initiate Th1 type immune response.

Antigen Presentation↗
PubMed1997

The use of cytokines in the treatment of solid tumours.

This manuscript reviews the clinical trials with cytokines in solid tumors. Starting with Interleukin 2 alone or in association with other cytokines and chemotherapy, the reviews opens the prospects of future direction for the use of cytokines as adjuvant therapy, for vaccination or to inhibit the growth activity of certain cytokines.

Animals↗
PubMed1997

IL-6 is a survival prognostic factor in renal cell carcinoma.

It has been reported that a high plasmatic concentration of interleukin-6 (IL-6) is correlated to a lack of response to immunotherapy in several malignancies, suggesting that IL-6 was either a marker of tumour aggressiveness or had only a predictive value of response to immunotherapy. To discriminate between these two possibilities, a retrospective study was performed in a series of 19 patients with metastatic renal cell carcinoma who did not respond to IL-2/IFNalpha/5-FU treatment. Serum levels of IL-6, C-reactive Protein (CRP), soluble IL-2-receptor (sIL-2R), M-CSF and neopterin were assayed before treatment. IL-6 showed a significant correlation with patients median survival time (P < 0.016), suggesting that serum concentration of IL-6 before treatment is a marker of tumour aggressiveness rather than a predictive parameter for an immunological response.

Adult↗
PubMed1997

Generation and characterization of a mouse single-chain antibody fragment specific for disialoganglioside (GD2).

Anti-disialoganglioside (GD2) monoclonal antibodies (MAbs) have been used in vivo for immunolocalization and in phase I and II trials to target disseminated neuroblastoma, the most common extracranial solid tumor in children. However, the efficacy of these first-generation MAbs is likely to be improved by using engineered anti-GD2 antibodies. The generation of single-chain antibody fragments (scFv) could be very helpful as these molecules can be further modified to produce recombinant molecules with pre-defined properties such as immunotoxins, chimeric, or bispecific antibodies. Thus, a scFv directed against GD2 (scFv 7A4) was cloned, sequenced, and expressed. Its binding properties were characterized and compared to that of the parental MAb 7A4. Nucleotide sequence analysis of the scFv 7A4 indicated that its VH region belongs to the V region IIID subgroup and the V kappa to the V region II subgroup. The scFv 7A4 bound to GD2+ neuroblastoma cell lines but not to GD2- cell lines or to GD2- cells isolated from peripheral blood. ELISA and thin-layer chromatography (TLC) indicated that it retained the anti-GD2 specificity, and exhibited a slight cross-reaction with GD3 as the parental MAb. This scFv makes it possible to develop new useful reagents through genetic engineering for adjuvant tumor therapy.

Amino Acid Sequence↗
PubMed1997

A new set of monoclonal antibodies against human Fc gamma RII (CD32) and Fc gamma RIII (CD16): characterization and use in various assays.

Four mouse anti-human Fc gamma RII (CD32) (6C4, 2B2, 3D3, 93.4) (IgG1, kappa) and one anti-human Fc gamma RIII (CD16) (7.5.4) IgG1, kappa) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, kappa), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both Fc gamma RIIa and Fc gamma RIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to Fc gamma RII. All three MAbs immunoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti Fc gamma RII MAb, 93.4, directed against the intracellular region of Fc gamma RIIa1/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both Fc gamma RIIIa and Fc gamma RIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-Fc gamma RIIa1/2)/6C4 and 3G8 (anti-Fc gamma RIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble Fc gamma RIIa1/2 and Fc gamma RIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-Fc gamma RII and of the anti-Fc gamma RIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-Fc gamma RII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM.

Animals↗