CD40.FasL inhibits human T cells: evidence for an auto-inhibitory loop-back mechanism.

A chimeric CD40.FasL (CD40-CD95L) protein was designed with the combined capacities to bind to two surface receptors on activated T cells, CD40 ligand (CD40L; CD154) and Fas receptor (CD95). CD40.FasL, once tethered to the cell surface via one of its ends, can transmit a signal via its other end. In principle, simultaneous triggering from both ends is possible, and thus there is the intriguing potential for 'auto-inhibition' if such dual triggering occurs on the same cell itself. Several lines of evidence support this mechanism: (i) CD40.FasL is cytotoxic to Fas receptor-positive cell lines of different cell lineages, (ii) CD40.FasL's function is potentiated when there is enforced expression of CD40L on target cells, (iii) CD40.FasL inhibition does not require intercellular contact, as demonstrated by soft agar clone formation and cell dilution analysis and (iv) introduction of exogenous CD40 into the system interferes with CD40.FasL inhibition. Taken together, these data are consistent with a 'loop-back' inhibitory mechanism within individual activated (CD40L and Fas receptor expressing) T cells causing suicide of these T cells. Significantly, this type of fusion protein provides a unique way to confine immunoinhibition to activated T cells.

CTLA-4.FasL inhibits allogeneic responses in vivo.

CTLA-4.Fas ligand (CTLA-4.FasL), a paradigmatic 'trans signal converter protein (TSCP)', can attach to APC (via CTLA-4 binding to B7) and direct intercellular inhibitory signals to responding T cells (via FasL binding to Fas receptor), converting an activating APC-to-T cell signal into an inhibitory one. Our previous studies established that CTLA-4.FasL inhibits human primary mixed lymphocyte reactions (MLR) and induces alloantigen-specific hyporesponsiveness ex vivo. The present study extends this to an in vivo context. Using splenocytes from MHC-mismatched C57BL/6 and Balb/c mice, we demonstrated that his(6)CTLA-4.FasL, effectively inhibits murine MLR. Moving in vivo, we demonstrated that subcutaneously administered his(6)CTLA-4.FasL modulates the in vivo response of infused allogeneic splenocytes. his(6)CTLA-4.FasL reduces the number of cells in each cell division, and increases the percentage of dead cells in each division. These findings are consistent with an antigen-induced cell death of the alloreactive cells, and bolsters recombinant TCSP promise as a therapeutic for transplantation diseases.

A rheostatic mechanism for T-cell inhibition based on elevation of activation thresholds.

The activation of discrete T-cell responses depends on the triggering of individualized threshold numbers of T-cell receptors (TCRs). The results of this study indicate that the lipocalin placental protein 14 (PP14), a T-cell inhibitor produced by cells of the reproductive and hematopoietic systems, mediates its anti-inflammatory activity by elevating the T-cell activation threshold, thereby rendering T cells less sensitive to stimulation. Significantly, the data demonstrate hierarchical sensitivity of selected cytokine responses to PP14-mediated inhibition, with the hierarchy reflecting their respective activation thresholds. These findings suggest a novel paradigm for immunoinhibition wherein negative regulators can finely tune, rather than inactivate, T-cell responses, and thereby skew the cytokine output of immunologic responses.

The role of radiographic phase-contrast imaging in the development of intracochlear electrode arrays.

OBJECTIVE: This study describes the application of a new radiographic imaging modality, phase-contrast radiography, to in vitro human temporal bone imaging and investigates its use in the development of new electrode arrays for cochlear implants. BACKGROUND: The development of perimodiolar electrode arrays for cochlear implants requires detailed information from postoperative radiologic assessment on the position of the array in relation to the cochlear structures. Current standard radiographic techniques provide only limited details. MATERIALS AND METHODS: Nucleus standard electrode arrays and perimodiolar Contour electrode arrays were implanted into the scala tympani of 11 human temporal bones. Both conventional and phase-contrast radiographs were taken of each temporal bone for comparative purposes. RESULTS: Phase-contrast imaging provides better visualization of anatomic details of the inner ear and of the structure of the intracochlear electrode array, and better definition of electrode location in relation to cochlear walls. CONCLUSION: Phase-contrast radiography offers significant improvement over conventional radiography in images of in vitro human temporal bones. It seems to be a valuable tool in the development of intracochlear electrode arrays and cochlear implant research. However, this new radiographic technique still requires certain computational and physics challenges to be addressed before its clinical use can be established.

Induction of antitumor immunity via intratumoral tetra-costimulator protein transfer.

Our group recently described a novel two-step Fc(gamma1) fusion protein transfer method, which entails the docking of Fc(gamma1) fusion proteins onto cells precoated with chemically palmitated protein A (pal-prot A). In the present study, we have adapted this protein transfer method, originally used in an ex vivo context, for in situ tumor cell engineering, and in so doing, we have evaluated its utility for the induction of antitumor immunity via combinatorial costimulator protein transfer on to tumor cell surfaces. The feasibility of "painting" cells with preformed conjugates of a murine B7-1 costimulator derivative, B7-1.Fc(gamma1), and pal-prot A in a single step was first established ex vivo. Next, B7-1.Fc(gamma1):pal-prot A transfer was accomplished in vivo by directly injecting the preformed conjugates into highly aggressive L5178Y-R lymphomas grown intradermally in syngeneic mice. The presence of cell surface-associated B7-1 epitopes on cells of the injected tumors was documented by flow cytometric analysis of cells recovered subsequently from the injected tumors. B7-1.Fc(gamma1), along with Fc(gamma1) fusion protein derivatives of three additional costimulators (Fc(gamma1).4-1BBL, CD48.Fc(gamma1), and Fc(gamma1).CD40L) geared toward a variety of immune effectors, were together preconjugated with pal-prot A and injected directly into tumor beds. Significantly, this "tetra-costimulator" combination, delivered intratumorally, induced complete tumor regression in approximately 45% of treated mice, whereas control injections of pal-prot A alone had no therapeutic effect. Furthermore, there was evidence for systemic antitumor immunity in that tumor-specific CTLs were detected in spleens recovered from cured mice, and these mice were uniformly protected against tumor rechallenge at distant tumor sites. Hence, combinatorial costimulator transfer, coupled to intratumoral delivery, may have special advantages for the induction of antitumor immunity.

Properties of exogenously added GPI-anchored proteins following their incorporation into cells.

Isolated glycosylphosphatidylinositol (GPI)-anchored proteins, when added to cells in vitro, incorporate into their surface membranes and, once incorporated, exert their native functions. Virtually any protein of interest, if expressed as a GPI-reanchored derivative, can be modified to acquire this capacity. Such transfer of proteins directly to cells, termed "protein engineering" or "painting" constitutes an alternative to conventional gene transfer for manipulating cell surface composition that has many potential applications. Previous studies with incorporated GPI-anchored proteins have focused almost entirely on their extracellular functions. In this study, biotinylated human erythrocyte (E(hu)) decay accelerating factor, E(hu) acetylcholinesterase, and GPI-reanchored murine B7-1 and B7-2 were used as GPI-anchored reporters to characterize their plasma membrane organization and cell signalling properties following addition to Hela or Chinese hamster ovary cells. For each reporter, three types of cell-association were documented; (1) nonphysiological attachment and/or incomplete insertion, (2) uncomplexed membrane integration, and (3) organization into TX-100-resistant microdomains. Transit from the first two compartments into the third, i.e., microdomains, progressed slowly, continuing even after 24 to 36 h and was associated with the acquisition of cell signalling capacity. All four reporters, incorporated in two different detergents, behaved similarly. When organized in microdomains, caveolin and other GPI proteins co-isolated with the incorporated reporter. These results have implications for protein engineering of cells in general, and in particular, for cells such as modified tumor cell immunogens administered to patients for therapeutic purposes.

Chronic electrical stimulation of the auditory nerve using high surface area (HiQ) platinum electrodes.

High surface area cochlear implant electrodes with much smaller geometric surface areas than current designs might be used in the future to increase the number of stimulating electrodes along the carrier. Potential problems with an increase in charge density for a common stimulus resulting from decreasing the geometric surface area would be reduced by the enlarged real surface area of such electrodes. Electrochemically modified (HiQ) platinum (Pt) electrodes, with a real surface area approximately 75 times greater than the current standard Pt electrodes of the same geometric size, had shown in vitro a low polarization (Z(pol)) and electrode impedance (Z(e)), as well as a low residual direct current (DC). In this study we examined the chronic performance of HiQ electrodes in cats, which were bilaterally implanted with a two-channel HiQ or standard Pt scala tympani electrode array and unilaterally stimulated for periods of up to 2390 h. Stimuli consisted of 50 micros/phase charge-balanced biphasic current pulses presented at 2000 pulses/s/channel with a 50% duty cycle. Electrode impedance (Z(e)), access resistance (R(a)) and polarization impedance (Z(pol)) were calculated from current and voltage measurements obtained periodically throughout the implantation period. Immediately following implantation HiQ electrodes showed a significantly smaller Z(pol), resulting in a reduced Z(e) (P<0.0001) compared to standard electrodes, while there was no significant difference between R(a) of both electrode designs (P=0.91). Subsequently, Z(e) generally increased mainly due to a rise in R(a), which dominated Z(e) and obliterated the effect of a lower Z(pol) on Z(e) in HiQ electrodes. Peak R(a) levels correlated closely (r=0.85) with the amount of intracochlear fibrous tissue found adjacent to the array. Following explantation of the array, voltage waveforms for both electrode designs recorded in saline were again very similar to those recorded immediately after implantation. Mean DC levels were consistently lower for HiQ electrodes compared with standard electrodes (22.45 nA vs 134.7 nA). Histopathological examination of corresponding cochlear sections comparing the stimulated test side with the unstimulated control side showed no significant difference (P>0.05) for either animals implanted with HiQ electrodes (n=6) or standard electrodes (n=2). Nor were there any significant differences between the spiral ganglion cell density of the basal turn implanted with HiQ or standard electrodes for both the stimulated test (P=0.31) and the unstimulated control side (P=0.84). Although these findings are based on a small group of animals implanted with standard electrodes (n=2), and those negative statistical results could potentially be due to the small sample size, similar spiral ganglion cell survival was found in a previous study of a larger group of animals using standard electrodes stimulated with the same stimulus paradigm as in the present study [Xu et al. (1997) Hear. Res. 105, 1-29]. Our data indicate that while some initial advantages of HiQ electrodes are lost during chronic implantation due to intracochlear fibrous tissue growth, low DC levels and the high surface area appear to be maintained, suggesting that HiQ electrodes may have important clinical applications.

The contour electrode array: safety study and initial patient trials of a new perimodiolar design.

OBJECTIVE: The aim of these studies was to investigate the insertion properties and safety of a new intracochlear perimodiolar electrode array design (Contour). BACKGROUND: An electrode array positioned close to the neural elements could be expected to reduce stimulation thresholds and might potentially reduce channel interaction. METHODS: Two sequential studies were conducted. In study 1, the Contour electrode array was inserted in 12 human temporal bones. After cochlear surface preparation, the position of the array was noted and the basilar membrane was examined for insertion damage. On the basis of the outcome of this temporal bone study, study 2 investigated the Contour array, mounted on a Nucleus CI-24 M device and implanted in three adult patients. RESULTS: Study I showed that in 10 temporal bones, the Contour array was positioned close to the modiolus, and the basilar membrane was intact. In the two remaining bones, the arrays had pierced the basilar membrane and were positioned in the scala vestibuli apical to the penetration. Statistical analysis showed an equivalent probability of insertion-induced damage of the two array designs. In study 2, image analysis indicated that the Contour electrodes were positioned closer to the modiolus than the standard Nucleus straight array. Lower T and C levels, but higher impedance values, were recorded from electrodes close to the modiolus. Initial speech perception data showed that all patients gained useful open-set speech perception, two patients achieving scores of 100% on sentence material 3 months postoperatively. CONCLUSIONS: The temporal bone studies showed the Contour electrode array to be generally positioned closer to the modiolus than the standard Nucleus straight array, and to have an equivalent probability of causing insertion-induced damage.

CTLA-4-Fas ligand functions as a trans signal converter protein in bridging antigen-presenting cells and T cells.

Co-stimulator blockade and trans inhibitory signaling, using agents such as CTLA-4-Ig and Fas ligand (FasL) respectively have been invoked as alternative strategies for suppressing pathogenic T cells. This study describes a novel hetero-bifunctional fusion protein, CTLA-4-FasL, designed to combine within a single protein both co-stimulator blocking and trans inhibitory signaling potentials. A chimeric expression cassette, in which the ectodomain coding sequences for CTLA-4 and FasL were linked in-frame, was used to produce a CTLA-4-FasL fusion protein. CTLA-4-FasL binding to both B7-1/B7-2-expressing Daudi B cells and Fas-expressing Jurkat T cells was documented by immunofluorescence and flow cytometry. The capacity of CTLA-4-FasL to induce apoptosis in Jurkat targets was markedly enhanced by the addition of Daudi and other B7-1/B7-2(+) B cell lines, which provided a membrane platform for the otherwise soluble CTLA-4-fusion protein. Moreover, in dual-chamber experiments, Daudi cells pre-coated with CTLA-4-FasL demonstrated Jurkat inhibitory activity that was cell-contact dependent. Significantly, when used to inhibit in vitro cellular proliferation of peripheral blood mononuclear cells, CTLA-4-FasL was approximately 1000-fold more potent than the extensively characterized CTLA-4-Ig fusion protein. Furthermore, the degree of inhibition induced by CTLA-4-FasL substantially surpassed that observed for CTLA-4-Ig and a soluble FasL when used in combination. CTLA-4-FasL represents the first of a novel class of fusion proteins, designated here as 'trans signal converter proteins', that combine trans signal masking and direct trans signaling functions.

Surgical implications of perimodiolar cochlear implant electrode design: avoiding intracochlear damage and scala vestibuli insertion.

OBJECTIVE: To review the mechanisms and nature of intracochlear damage associated with cochlear implant electrode array insertion, in particular, the various perimodiolar electrode designs. Make recommendations regarding surgical techniques for the Nucleus Contour electrode to ensure correct position and minimal insertion trauma. BACKGROUND: The potential advantages of increased modiolar proximity of intracochlear multichannel electrode arrays are a reduction in stimulation thresholds, an increase in dynamic range and more localized neural excitation. This may improve speech perception and reduce power consumption. These advantages may be negated if increased intracochlear damage results from the method used to position the electrodes close to the modiolus. METHOD: A review of the University of Melbourne Department of Otolaryngology experience with temporal bone safety studies using the Nucleus standard straight electrode array and a variety of perimodiolar electrode array designs; comparison with temporal bone insertion studies from other centres and postmortem histopathology studies reported in the literature. Review of our initial clinical experience using the Nucleus Contour electrode array. RESULTS: The nature of intracochlear damage resulting from electrode insertion trauma ranges from minor, localized, spiral ligament tear to diffuse organ of Corti disruption and osseous spiral lamina fracture. The type of damage depends on the mechanical characteristics of the electrode array, the stiffness, curvature and size of the electrode in relation to the scala, and the surgical technique. The narrow, flexible, straight arrays are the least traumatic. Pre-curved or stiffer arrays are associated with an incidence of basilar membrane perforation. The cochleostomy must be correctly sited in relation to the round window to ensure scala tympani insertion. A cochleostomy anterior to the round window rather than inferior may lead to scala media or scala vestibuli insertion. CONCLUSION: Proximity of electrodes to the modiolus can be achieved without intracochlear damage provided the electrode array is a free fit within the scala, of appropriate size and shape, and accurate scala tympani insertion is performed.

alpha2-macroglobulin modulates the immunoregulatory function of the lipocalin placental protein 14.

Human placental protein 14 (PP14; also known as glycodelin and progesterone-associated endometrial protein) is an immunosuppressive protein of the lipocalin structural superfamily. Mechanisms regulating serum PP14's immunosuppressive activity remain to be elucidated. In the present study, an interaction between PP14 and a major serum protein carrier, alpha(2)-macroglobulin (alpha(2)M), was documented for the first time. Using native gel electrophoresis, we showed that PP14, as well as its alternative splice variant PP14.2, binds to both alpha(2)M and methylamine-activated (MA)-alpha(2)M. Cross-competition studies demonstrated that the variants compete for binding to alpha(2)M. PP14 bound to alpha(2)M and MA-alpha(2)M with K(d) values of 167+/-70 and 221+/-56 nM (means+/-S.D.) respectively, as determined by surface plasmon resonance. Significantly, the addition of alpha(2)M or MA-alpha(2)M to a T-cell proliferation assay strongly potentiated the inhibitory capacity of PP14. On the basis of these findings, alpha(2)M emerges as the first serum protein that can physically associate with, and thereby regulate, PP14. Moreover, this represents the first documented interaction between the protein carrier alpha(2)M and a lipocalin protein.

Glycosyl-phosphatidylinositol reanchoring unmasks distinct antigen-presenting pathways for CD1b and CD1c.

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b. DAF and CD1c. DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c. DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b. DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c. DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b. DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.

Comparison of electrode position in the human cochlea using various perimodiolar electrode arrays.

OBJECTIVE: This study was conducted to evaluate the insertion properties and intracochlear trajectories of three perimodiolar electrode array designs and to compare these designs with the standard Cochlear/Melbourne array. BACKGROUND: Advantages to be expected of a perimodiolar electrode array include both a reduction in stimulus thresholds and an increase in dynamic range, resulting in a more localized stimulation pattern of the spiral ganglion cells, reduced power consumption, and, therefore, longer speech processor battery life. METHODS: The test arrays were implanted into human temporal bones. Image analysis was performed on a radiograph taken after the insertion. The cochleas were then histologically processed with the electrode array in situ, and the resulting sections were subsequently assessed for position of the electrode array as well as insertion-related intracochlear damage. RESULTS: All perimodiolar electrode arrays were inserted deeper and showed trajectories that were generally closer to the modiolus compared with the standard electrode array. However, although the precurved array designs did not show significant insertion trauma, the method of insertion needed improvement. After insertion of the straight electrode array with positioner, signs of severe insertion trauma in the majority of implanted cochleas were found. CONCLUSIONS: Although it was possible to position the electrode arrays close to the modiolus, none of the three perimodiolar designs investigated fulfilled satisfactorily all three criteria of being easy, safe, and atraumatic to implant.

Hierarchical costimulator thresholds for distinct immune responses: application of a novel two-step Fc fusion protein transfer method.

Activation of T cells is dependent upon coordinate engagement of Ag and costimulator receptors on their surfaces. In the case of the Ag receptors (TCRs), activation thresholds have been defined, with the number of TCRs that must be triggered to stimulate cytokine secretion by individual activated T cells differing for the various cytokines. In the present study, we have determined whether comparable activation thresholds exist for the costimulator receptors on T cells. To facilitate this type of quantitative costimulator analysis, we developed a novel two-step protein transfer approach that permits delivery of graded amounts of proteins to APC surfaces. By adding a human B7-1. Fcgamma1 (Fc domain of human IgG1) fusion protein to cells precoated with palmitated protein A, fine titration of the B7-1 extracellular domain was achieved. The B7-1. Fcgamma1 reincorporated into cell membranes by this method retained costimulator function, as measured by an in vitro proliferation assay. The degree of proliferation was dependent on the surface density of B7-1. Fcgamma1. Significantly, the threshold B7-1. Fcgamma1 density required for cytokine production differed between IFN-gamma and IL-2 and mirrored the hierarchy (IFN-gamma < IL-2) described previously for the TCR activation threshold. Hence, this study invokes a novel protein transfer strategy to establish that the levels of surface costimulator on APCs can dictate both the magnitude and the quality of evoked T cell responses. The notion of costimulator receptor activation thresholds emerges.

The imprinted H19 gene is a marker of early recurrence in human bladder carcinoma.

AIMS: To investigate the expression of the imprinted oncofetal H19 gene in human bladder carcinoma and to examine the possibility of using it as a tumour marker, similar to other oncofetal gene products. METHODS: In situ hybridisation for H19 RNA was performed on 61 first biopsies of bladder carcinoma from Hadassah Medical Centre in Jerusalem. The intensity of the reaction and the number of tumour cells expressing H19 in each biopsy were evaluated in 56 patients, excluding biopsies with carcinoma in situ. The medical files were searched for demographic data and disease free survival. RESULTS: More than 5% of cells expressed H19 in 47 of the 56 (84%) biopsies. There was a decrease in the number of cells expressing H19 with increasing tumour grade (loss of differentiation) (p = 0.03). Disease free survival from the first biopsy to first recurrence was significantly shorter in patients with tumours having a larger fraction of H19 expressing cells, controlling for tumour grade. This was also supported by the selective analysis of tumour recurrence in patients with grade I tumours. CONCLUSIONS: It might be possible to use H19 as a prognostic tumour marker for the early recurrence of bladder cancer. In addition, for the gene therapy of bladder carcinoma that is based on the transcriptional regulatory sequences of H19, the expression of H19 in an individual biopsy could be considered a predictive tumour marker for selecting those patients who would benefit from this form of treatment.

Characterization of human and mouse H19 regulatory sequences.

H19 is expressed in a large percentage of bladder tumors, but not expressed in healthy bladder tissue. The aim of this study is to define H19 optimal transcriptional regulatory sequences in tumor cells, which can potentially be used to control expression of a toxin gene in constructs to be used in bladder cancer gene therapy trials in mice and human. Transient expression assays revealed that elements responsible for promoter activity are contained within the 85 bp upstream region. The transcriptional activity of this region was strongly inhibited by the methylation of the Hpa II sites. A modest cell specificity is conferred by the upstream sequences. The human and murine promoter activities were significantly increased by the human H19 4.1 kb enhancer sequence. The 85 bp H19 upstream region contains all the elements to interact with the enhancer. We showed that the human H19 promoter is highly active in a murine bladder carcinoma cell line, justifying its use to drive the expression of a cytotoxin gene in gene therapy trials in mice.

Protein transfer of glycosyl-phosphatidylinositol (GPI)-modified murine B7-1 and B7-2 costimulators.

The feasibility of using protein transfer as a means for enhancing the immunogenicity of murine tumor cells was evaluated. Glycosyl-phosphatidylinositol (GPI)-modified variants of the murine costimulators B7-1 (CD80) and B7-2 (CD86), designated B7-1.GPI and B7-2.GPI, respectively, were immunoaffinity-purified from CHO-K1 cells transfected with glutamine synthetase amplification/expression constructs encoding each of these chimeric proteins. The proteins, once purified in detergent-depleted pseudomicelles, were exogenously incorporated into the membranes of several different murine tumor lines (EL-4, SMUCC-1, BW5147.3, P815, Ag104A, and EMT6). Successful membrane painting with the B7.GPI proteins was documented by immunofluorescence and flow cytometry, and membrane integration was verified by demonstrating that the reincorporated proteins were phosphatidylinositol-phospholipase C-sensitive, glycosyl-phosphatidylinositol-phospholipase D-resistant, and refractory to removal with dimyristylphosphatidylcholine vesicles. Significantly, B7-1.GPI and B7-2.GPI could be together copainted onto EL-4 cell surfaces with no interference observed between the two. A standard in vitro proliferation assay was used to show that both of the B7.GPI proteins retained costimulator function after membrane reincorporation. These findings further validate the therapeutic potential of protein-transferred costimulator.GPIs and pave the way for their combinatorial use in animal tumor models.

The expansion of human gammadelta T cells in response to Daudi cells requires the participation of CD4+ T cells.

The Burkitt's lymphoma cell line Daudi is a potent inducer of human gammadelta T-cell expansion. Using an in vitro culture system comprised of irradiated Daudi cells as stimulators and normal human lymphocytes as responders, the cellular determinants of this response were investigated. Three of four monoclonal antibodies (mAbs 1-1C4, L243, and 9.3F10) directed against disparate epitopes of human major histocompatibility complex (MHC) class II, as well as a mAb with specificity for CD4 (OKT4), inhibited the expansion of gammadelta T cells in response to Daudi cell stimulators. mAbs with a specificity for CD74 and CD8 were non-inhibitory. Lymphocyte depletion experiments demonstrated a critical role for the CD4+ T-cell subset in the expansion of gammadelta T cells. Other data pointed towards requirements for direct cell contact in this system, and the addition of exogenous recombinant interleukin (IL)-2, IL-4, and IL-12 failed to reconstitute gammadelta T-cell expansion in CD4+ lymphocyte-depleted cultures. These results complement previous findings in murine infectious disease and mycobacterial systems, providing a direct demonstration that CD4+ T cells play a role in gammadelta T-cell expansion through an interaction with human leucocyte antigen (HLA) class II on Daudi cells. The data point towards important functional links between the acquired and natural immune systems.

Expression of the imprinted H19 oncofetal RNA in epithelial ovarian cancer.

STUDY: To examine the expression of the imprinted maternally expressed H19 gene in benign, low malignant potential (borderline) and malignant surface epithelial ovarian tumors. DESIGN: In situ hybridization for H19 RNA using S-labeled and digoxigenin-labeled probes was performed on paraffin sections of ovarian surface epithelial tumors. The serous tumors included nine section cystadenomas, twelve serous tumors of low malignant potential and twenty serous carcinomas, grade I-IIII (FIGO classification). A smaller group included two mucinous cystadenomas, four mucinous tumors of low malignant potential and two mucinous cystadenocarcinomas. RESULTS: H19 expression was found to be positive in 6/9 (67%) serous cystadenomas, 9/12 (75%) of serous tumors of low malignant potential and 13/20 (65%) of invasive serous carcinomas. Expression in mucinous tumors was confined to the stroma beneath the epithelial lining. CONCLUSION: H19 is expressed in the majority of serous epithelial tumors. Taking into consideration the high percentage of H19 expressing serous ovarian neoplasms we suggest that H19 RNA may be used as an adjuvant tumor marker for the diagnosis and mainly for staging and follow-up of patients with serous ovarian carcinoma.