Development of standardized cell culture conditions for tumor cells with potential clinical application.

BACKGROUND: Tumor cell lines have enormous value for the study of different aspects of cancer biology and have also recently gained great importance in autologous cell-based anti-tumor therapies. However, the use of these cells is still limited because in vitro growth is hampered by suboptimal culture conditions and current media contain fetal bovine serum (FBS), which poses serious safety concerns regarding clinical application. METHODS: To address this drawback, we aimed to develop a strategy for optimization of the culture medium for human medullary thyroid carcinoma (MTC) cell lines as a model system. We combined the general cell screening system (GCSS), which continuously measured the growth behavior of cells in a 96-well plate format, with statistically based experimental designs. RESULTS: The results obtained clearly demonstrated that, just by changing the composition of the basal medium, a significantly enhanced growth rate could be observed, and by subsequent addition of several substances a serum-free cell culture medium could be developed. This medium allowed the propagation of two MTC cell lines comparable with conventionally used serum-supplemented medium. DISCUSSION: We present a fast and easy way to screen for substances that are essential for tumor cell growth in vitro. Furthermore, these tumor cells can be adapted to culture conditions that allow the use of the cells in safe cell-based therapies. This is of utmost importance because of increasing regulatory requirements.

Intracellular concentration of the HIV protease inhibitors indinavir and saquinavir in human endothelial cells.

Human vascular endothelial cells may serve as targets and a reservoir for human immunodeficiency virus type 1 (HIV-1). The antiviral activity of HIV protease inhibitors is reported to be related directly to the intracellular amount of the drug. To assess intracellular concentrations of two HIV protease inhibitors, human umbilical venous endothelial cells (HUVECs) were exposed for 3 h and 24 h to 100, 10 and 1 mg/L indinavir and saquinavir. Intracellular drug concentrations and the total drug amount in the supernatant were measured by means of high-performance liquid chromatography (HPLC). Exposure of HUVECs to 10 and 1 mg/L indinavir and saquinavir resulted in undetectable intracellular drug levels in 6 x 10(5) cells/well. Incubation of cells with solutions of 100 mg/L indinavir and saquinavir led to mean intracellular concentrations of indinavir (132 +/- 56 mg/L after 3 h and 150 +/- 29 mg/L after 24 h, respectively) and of saquinavir (96 +/- 10 mg/L after 3 h and 100 +/- 5 mg/L after 24 h) that were comparable to the levels determined for the substances in the supernatant over time (P > 0.001). These data indicate that intracellular concentrations of indinavir and saquinavir correlate well with the extracellular levels. Consequently, measurements of drug concentrations in patient's plasma by HPLC are assumed to be a good means of monitoring the intracellular drug concentration.

A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1.

We have established a panel of human monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1). The antibodies 2F5 and 2G12 have been identified to be two of the most potently in vitro neutralizing antibodies against HIV-1. Here we report on a further antibody, 4E10, of similar in vitro neutralizing potency. 4E10 binds to a novel epitope C terminal of the ELDKWA sequence recognized by 2F5, which has been so far the only described broadly neutralizing anti-gp41 antibody. Both 4E10 and 2F5 bind only weakly to infected cells compared with gp120-specific 2G12 and polyclonal anti-HIV-1 immunoglobulin (HIVIG), but show potent in vitro neutralizing properties. 4E10 neutralizes potently not only tissue culture-adapted strains but also primary isolates of different clades, including A, B, C, D, and E. Viruses that were found to be resistant to 2F5 were neutralized by 4E10 and vice versa; none of the tested isolates was resistant to both anti-gp41 antibodies. This confirms that the region recognized by 2F5 and 4E10 is essential for viral infectivity and may be important for vaccine design. Moreover, our results suggest that 4E10 should be further investigated for passive anti-HIV immunotherapy.

B cell-mediated infection of stimulated and unstimulated autologous T lymphocytes with HIV-1: role of complement.

In vivo, human immunodeficiency virus type 1 (HIV-1) is opsonized with complement fragments and virus-specific antibodies (Ab). Thus, HIV is able to interact with complement receptor (CR) - and Fc receptor (FcR) - positive cells such as B cells, follicular dendritic cells or macrophages. In this study we demonstrate that the interaction between B cells and HIV has an impact on autologous primary T cell infection in vitro. We confirmed the presence of complement-fragments and virus-specific Ab on serum-treated HIV using a virus-capture assay. In experiments with CR2-specific Ab we showed that the virus/B cell interaction was mainly dependent on CR2. In infection experiments immobilisation of HIV on stimulated tonsil B cells greatly enhanced the infection of interleukin (IL)-2-activated autologous tonsil T cells. Surprisingly, enhancement of T cell infection by B cell-HIV complexes was observed even in the absence of mitogenic stimuli such as PMA and was independent of the addition of exogenous IL-2. Taken together, these results indicate that primary B cells are able to efficiently transmit opsonised HIV to autologous primary T cells and induce a massive enhancement of infection. These in vitro experiments mimic the in vivo situation in the lymphoid tissue and suggest an alternative mechanism for the infection of primary T cells.

Detachment of human immunodeficiency virus type 1 from germinal centers by blocking complement receptor type 2.

After the transition from the acute to the chronic phase of human immunodeficiency virus (HIV) infection, complement mediates long-term storage of virions in germinal centers (GC) of lymphoid tissue. The contribution of particular complement receptors (CRs) to virus trapping in GC was studied on tonsillar specimens from HIV-infected individuals. CR2 (CD21) was identified as the main binding site for HIV in GC. Monoclonal antibodies (MAb) blocking the CR2-C3d interaction were shown to detach 62 to 77% of HIV type 1 from tonsillar cells of an individual in the presymptomatic stage. Although they did so at a lower efficiency, these antibodies were able to remove HIV from tonsillar cells of patients under highly active antiretroviral therapy, suggesting that the C3d-CR2 interaction remains a primary entrapment mechanism in treated patients as well. In contrast, removal of HIV was not observed with MAb blocking CR1 or CR3. Thus, targeting CR2 may facilitate new approaches toward a reduction of residual virus in GC.

A simple method to quantify staphylococcal protein A in the presence of human or animal IgG in various samples.

Immunoassays designed to measure low concentrations of staphylococcal protein A (SPA) that have been leached into antibody preparations intended for therapeutic use are subject to differing degrees of interference. Methods established to quantify SPA in murine antibody preparations are not accurate in the presence of human or humanized IgG. We report the development of an enzyme-linked immunosorbent assay (ELISA) for SPA with a detection limit of 7 pg/ml and the optimization of a method that permits complete dissociation of SPA-immunoglobulin-complexes. This assay is a modification of our heat-mediated dissociation (HD-SD) treatment with sodium dodecyl sulfate (SDS) and diethylenetriaminepentacetic acid (DTPA) for total immune-complex dissociation, in which the heat treatment has been prolonged and the diluent is characterized by increased protein content and buffering capacity. The diluent developed contains SDS, DTPA and bovine serum albumin dissolved in a 0.1 M phosphate buffer (pH 7.2). To validate the efficiency of this novel method, a series of samples have been assayed, including samples reconstituted in vitro, samples of purified antibodies, and plasma from patients. The described method has been shown to be generally efficient in quantitating all native and recombinant SPA in samples containing up to 50 mg/ml of human IgG. These data demonstrate the utility of this technique in determining SPA contamination of recombinant immunoglobulin therapeutic products.

Human immunodeficiency virus type 1 derived from cocultures of immature dendritic cells with autologous T cells carries T-cell-specific molecules on its surface and is highly infectious.

During the budding process, human immunodeficiency virus type 1 (HIV-1) acquires cell surface molecules; thus, the viral surface of HIV-1 reflects the antigenic pattern of the host cell. To determine the source of HIV-1 released from cocultures of dendritic cells (DC) with T cells, immature DC (imDC), mature DC (mDC), T cells, and their cocultures were infected with different HIV-1 isolates. The macrophage-tropic HIV-1 isolate Ba-L allowed viral replication in both imDC and mDC, whereas the T-cell-line-tropic primary isolate PI21 replicated in mDC only. By a virus capture assay, HIV-1 was shown to carry a T-cell- or DC-specific cell surface pattern after production by T cells or DC, respectively. Upon cocultivation of HIV-1-pulsed DC with T cells, HIV-1 exclusively displayed a typical T-cell pattern. Additionally, functional analysis revealed that HIV-1 released from imDC-T-cell cocultures was more infectious than HIV-1 derived from mDC-T-cell cocultures and from cultures of DC, T cells, or peripheral blood mononuclear cells alone. Therefore, we conclude that the interaction of HIV-1-pulsed imDC with T cells in vivo might generate highly infectious virus which primarily originates from T cells.

A simple and robust method for the complete dissociation of HIV-1 p24 and other antigens from immune complexes in serum and plasma samples.

Accuracy of antigen determination in human plasma samples is often adversely affected by immune complex formation between antigens (e.g., HIV-1 p24 protein) and specific antibodies. In this study we describe an optimized method for complete immune complex dissociation (ICD) in plasma. This method is based on heat denaturation of antibodies and utilizes a defined solution of sodium dodecyl sulfate (SDS) and diethylenetriaminepentaacetic acid (DTPA) as diluent. The efficiency of this procedure for ICD was compared with those of published methods, employing heat denaturation alone and acidification. Plasma samples from patients participating in anti-retroviral treatments and samples reconstituted in vitro were treated and analyzed in parallel. HIV-1 p24 antigen was determined by quantitative enzyme-linked immunosorbent assay (ELISA). In 312 samples from 97 patients, antigenemia was found in 44.9% when measured directly and in 87.2% after this treatment. In a subset of 56 samples, 21.4% tested positive prior to treatment, while after either novel treatment, heat denaturation or acidification, these samples tested positive in 80.4%, 62.5% and 60.7%, respectively. In 94% of cases viral RNA was detected. This improved procedure for ICD provides a reliable and convenient method for complete and accurate p24 antigen detection in human plasma and is applicable to commercially available test kits.

Dendritic cells transmit human immunodeficiency virus type 1 to monocytes and monocyte-derived macrophages.

Previous studies have shown that human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DC) to replicate and spread among CD4(+) T cells. To explain the predominance of non-syncytium-inducing (NSI) over syncytium-inducing (SI) strains during the initial viremia of HIV, we investigated the ability of blood monocyte (Mo)-derived DC to transmit HIV-1 to CD4(+) cells of the monocytoid lineage. First, we demonstrate that in our system, DC are able to transmit NSI strains, but not SI strains, of HIV-1 to fresh blood Mo and to Mo-derived macrophages (MDM). To establish a productive infection, a 10-fold-lower amount of virus was necessary for DC-mediated transmission of HIV-1 to Mo than in case of cell-free infection. Second, immature CD83(-) DC (imDC) transmit virus to Mo and MDM with higher efficacy compared to mature CD83(+) DC (maDC); this finding is in contrast to data previously obtained with CD4(+) T cells. Third, maturation from imDC to maDC efficiently silenced expression of beta2-integrins CD11b, CD11c, and CD18 by maDC. Moreover, monoclonal antibody against CD18 inhibited transmission of HIV-1 from imDC to Mo. We propose that the adhesion molecules of the CD11/CD18 family, involved in cell-cell interactions of DC with the microenvironment, may play a major role in imDC-mediated HIV-1 infection of Mo and MDM.

Interferon alpha primes early proliferative response of bone marrow cells in vivo.

Using colony-forming assays, a number of previous studies established that interferon alpha (IFN-alpha) could cause bone marrow cell (BMC) suppression. In this study, the suppressive effect of IFN-alpha is however shown to be time-dependent, occurring only 7-8 days after transfer of BMC obtained from IFN-alpha-treated mice to growth factor-containing culture medium. In contrast, in the interval before suppression is observed, BMC obtained from IFN-alpha-treated mice initially proliferated more rapidly than BMC from placebo-treated mice. These findings suggest that IFN-alpha acts in vivo to prime the proliferative responses of BMC, a hitherto unexpected action which may have clinical relevance.

Acquisition of host cell-surface-derived molecules by HIV-1.

OBJECTIVE: To determine the acquisition of host cell-membrane-derived molecules by HIV-1 during the budding process, and to investigate whether the uptake of these molecules is cell-type-specific and selective. DESIGN: Virions, propagated by four different cell types were analysed for the presence of adhesion molecules, glycosylphosphatidylinositol (GPI)-anchored proteins and various cell-surface markers. The pattern was compared with the phenotype of the HIV-1-infected cell. METHODS: For phenotypic analysis of virions a two-step assay was used. In the first step, virus was captured with monoclonal antibodies (in some cases polyclonal sera) against different cell-membrane proteins. In a second step, the presence of virus was measured by determining the concentration of the virus-specific p24 core antigen. The expression of surface molecules on uninfected and HIV-1IIIB-infected cells was analysed by FACS. RESULTS: Depending on the cell type used for virus propagation, different cell-membrane molecules were found on the virus surface reflecting the corresponding cell type. The uptake of these molecules was selective to a certain degree. No CD4 and CD87 molecules were detectable on HIV-1, although both molecules were present on uninfected and HIV-1-infected cells. CR3 and CDw108 could not be seen on uninfected cells, but wre detectable on infected cells and virions. CONCLUSIONS: During the budding process HIV-1 acquires a variety of cell-type-specific cell-surface molecules. Certain cell-membrane molecules become upregulated during HIV-1-infection and are then found on virions, whereas other molecules remain on the cell surface and do not become incorporated.

Cytokine activity assay by means of proliferation measured in plane convex microtiter wells.

A new assay to measure cell proliferation by turbidimetric evaluation of cultures in specially designed microtiter plates was set up. The IL-2-dependent proliferation of CTLL-2 cell line was used as a model. The novel microtiter plate detection system, the General Cell Screening System (GCSS) was used for the evaluation of the cell proliferation assay. The test system utilizes the population growth rate (mu) of the cells. The tests were performed in specially designed microtiter plates which were read in a scanning spectrophotometer. These results were compared with colorimetric assays, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) as substrates. In contrast to conventional methods, the presently described system is not an end-point method, this means that cell growth can be observed over a certain period and is not limited to a single moment during the period of cell growth. Another advantage is the reduction of manual manipulations and the avoidance of toxic or radioactive substances and organic solvents. Therefore, a higher number of samples can be analyzed compared to other methods. The range for detection was 0.015-10 units IL-2 per well, and the accuracy is in the range of < 0.005 standard deviation.

Human interleukin-2 (IL-2) expressed by transfected mammalian cells.

cDNA for human interleukin-2 (IL-2) was cloned into the pRc/RSV vector for expression in animal cells. Baby hamster kidney (BHK-21) cells and Chinese hamster ovary (CHO) cells were transfected several times using calcium phosphate and electroporation methods with the construct pRc/RSV SIGIL2. Different transfection efficiencies were obtained. The biological test on CTLL-2 (mouse cytotoxic lymphocytes) showed that the kinetics of cell proliferation were different from those of rIL-2 (recombinant IL-2) expressed in bacteria and in BHK cells. When high concentrations of rIL-2 were applied, an inhibitory effect on CTLL-2 was observed when bacterial product was used, whilst rBHK interleukin caused no inhibition. Recombinant BHK IL2 induced a slower response of CTLL-2 cells at the beginning of the cultivation, however, prolonged activity was detected at the later stage of the experiment.

A broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1.

We have established a hybridoma clone, designated 2F5, secreting a neutralizing human monoclonal antibody (MAb) specific for gp41 of human immunodeficiency virus type 1 (HIV-1). The epitope of MAb 2F5 was mapped to amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala on the ectodomain of gp41. In this study different in vitro test systems were used to characterize the neutralizing properties of MAb 2F5. In syncytium inhibition assays, fusion inhibition experiments, and neutralization assays on different HIV-susceptible cells (H9, U937, and peripheral blood mononuclear cells) MAb 2F5 showed broad-spectrum neutralizing capacity against HIV-1 laboratory isolates IIIB, MN, RF, and SF2. In addition, primary isolates from AIDS patients were also neutralized.

HIV-1 gp41 binding proteins and antibodies to gp41 could inhibit enhancement of human Raji cell MHC class I and II expression by gp41.

Based on our findings, that HIV-1 soluble gp41 could bind to several proteins on the human, T, B and monocyte cells independently of CD4, we examined the effect of HIV-1 soluble gp41 (sgp41;Env amino acids 539-684) on surface expression of MHC I and II, ICAM-1 and CD21 molecules on human Raji cells. Flow cytometry (FACS) analysis demonstrated that sgp41 could selectively enhance MHC class I and II expression on Raji cells, but did not increase expression of other cell surface antigens, such as, CD21 and CD54 (ICAM-1). Soluble gp41 could also enhance MHC class I and II expression on another human B cell line, Bjab. The sgp41-dependent enhancement of the MHC class I and II expression on Raji cells is time- and dose-dependent. The sgp41 enhancement effect on the MHC antigen expression could be inhibited by the sgp41-binding proteins of 45, 49 and 62 kD (isolated from Raji-lysate) which could inhibit the spg41-binding to Raji cells. Interestingly, this sgp41-dependent enhancement of the MHC class I and II expression could also be inhibited by two mAbs to HIV-1 gp41, but not by a third mAb binding to a different site on gp41. These results demonstrate that HIV-1 sgp41 can selectively enhance the human Raji cell MHC class I and II antigen expression and this enhancement effect could be inhibited by the sgp41-binding proteins and anti-gp41 antibodies, and suggest that the sgp41-dependent enhancement is mediated by its binding to Raji membrane proteins of 45, 49 and 62 kD.

HIV-1 gp41 enhances major histocompatibility complex class I and ICAM-1 expression on H9 and U937 cells.

Based on our findings that HIV-1 (human immunodeficiency virus type 1) soluble gp41 (sgp41; amino acids 539-684) bound to human T, B, and monocyte cells and enhanced major histocompatibility complex (MHC) class I and II antigen expression on Raji cells, we examined the effect of HIV-1 sgp41 on the surface expression of MHC I and II, ICAM-1, and CD4 molecules on human H9 and U937 cells. Flow cytometry (FACS) analysis demonstrated that sgp41 selectively enhanced MHC class I expression by about 75% on H9 cells and by about 85% on U937 cells, while the ICAM-1 expression was increased by about 70% only on H9 cells and remained unaltered on U937 cells; other molecules, such as MHC class II and CD4, remained unaltered. By comparison, alpha-, beta-, and omega-interferons, but not gamma-interferon, showed similar effects as sgp41 on the expression of MHC class I and ICAM-1 on H9 and U937 cells. The results suggest that HIV-1 gp41 may have a biological function that is involved in the regulation of human MHC class I and ICAM-1 expression.

Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS.

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.

HIV-1 gp41 binds to two proteins in cell culture supernatant of human B cell line Raji and monocyte cell line U937.

Based on our findings that HIV-1 gp41 can bind independently of CD4 to the human T cell line H9, B cell line Raji and monocytic cell line U937, as well as human peripheral blood mononuclear cells preferentially to B lymphocytes and monocytes, we examined whether soluble protein for HIV-1 gp41 binding exists in the cell culture supernatant of Raji and U937. Using HIV-1 recombinant soluble gp41 (sgp41; Env amino acid 539-684) attached to sepharose beads, the cell culture supernatants of Raji and U937 were absorbed. By SDS-PAGE of sgp41-eluates of these cell culture supernatants three protein bands of 70, 75 and 92kD were stained with Coomassie blue. By Western blot (ligand blot) analysis using sgp41, two protein bands of 70 and 75kD were observed in sgp41-eluates from Raji and U937 cell culture supernatants. These sgp41-eluates could inhibit the sgp41 binding to Raji and U937 cells. Our data indicate that Raji and U937 cells produce two soluble binding proteins for HIV-1 gp41.

Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization.

Electrofusion and EBV transformation were studied by immortalizing human PBLs from blood of HIV-1-positive volunteers. A panel of 33 cell lines producing human monoclonal antibodies (Hu-MAbs) against HIV-1 was established by cell fusion or EBV transformation. For the first fusion experiments the source of B lymphocytes was peripheral blood of HIV-1-infected donors in CDC stages II or III with CD4 cell counts higher than 500/mm3. Later on, from these patients only, those with high anti-HIV titers were chosen as blood donors. By that means the yield of stable specific hybridomas was increased twofold. In our experiments electrofusion turned out to be a more efficient immortalization method than EBV transformation, due to a high and constant immortalization rate. The hybridomas were stable after intensive subcloning and could be cultivated over a period of 8 months without loss in monoclonal antibody production. Immunoglobulin class, subtype, reactivity against HIV-1 proteins, Western blot patterns, immunofluorescence, and epitopes were characterized. The subtype of all antibodies was IgG1 or IgG3. The light chain was predominantly kappa. All antibodies showed reactivity against HIV-1 envelope or core protein. All hybridomas were stable and suited for mass production. Several Hu-MAbs are becoming an important tool in the field of diagnosis, research, and immunotherapy.