Radiologic diagnosis of abdominal trauma

We have characterized 10 monoclonal antibodies (Mabs) to recombinant murine thyrotropin receptor extracellular domain (mTSHR-ecd). Affinity purified mTSHR-ecd (amino acids 22-415), expressed in a baculovirus-insect cell system, was refolded in vitro and used to hyperimmunize female Balb/c mice. Spleens were removed 10 days after a final boost of 25 microg mTSHR-ecd intraperitoneally and intravenously, and the cells were fused to SP-2 cells and cloned. Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) with folded mTSHR-ecd antigen. Ten of 18 higher affinity hybridomas were selected at random and ascites fluids prepared. Nine of the monoclonals were of IgG 1 isotype, and one was IgM. Five Mabs (M3, M4, M5, M6, and M9) inhibited the binding of 125I-TSH to functional hTSHR expressed on Chinese hamster ovary (CHO) cells, and four (M1, M3, M5, and M9) blocked the TSH-stimulated generation of cyclic adenosine monophosphate (cAMP), using the same cells. The remaining Mabs appeared to be neutral in their interaction with native TSHR. The Mabs were also compared for their reactivity to mTSHR-ecd under folding (ELISA) and unfolding (reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) conditions. Most Mabs demonstrated reactivity to both conformational (folded) and linear (unfolded) forms of mTSHR-ecd, suggesting that they were generated primarily against linear epitopes although one Mab (M4) showed affinity for only folded antigen indicating a preference for a conformational epitope. Mapping the Mab epitopes using 26 overlapping peptides spanning the human (h)TSHR-ecd showed that 6 bound peptide 397-415, 1 bound peptide 352-371, and 1 peptide 22-41. These epitope mapped Mabs to the mTSHR-ecd, both receptor blocking and receptor neutral, will provide further insight into the structure-function of the TSHR ectodomain.     

Albumin-binding surfaces: in vitro activity

Immobilized monoclonal antibodies (Mabs) have been used to attract specific molecules to a solid surface from complex mixtures such as blood, plasma or serum, thereby directing the response to the modified substrate, a key goal in rational biomaterial design. The nature of the Mab dictated the nature of the response: anti-albumin antibodies were used to prevent cell and platelet adhesion in vitro, whilst anti-fibronectin Mabs promoted attachment. Patterned surfaces could be formed, bearing Mabs that generated adhesive and non-adhesive regions. Fibrinogen adsorption from plasma showed a Vroman peak on unmodified control polymer, which was reduced by 64% in the presence of surface-bound anti-albumin Mab. Immobilization of a control Mab reduced fibrinogen adsorption only slightly, implying an albumin-mediated effect. In static tests, platelet adhesion from human platelet rich plasma was significantly reduced by the immobilization of anti-HSA Mab when compared to the untreated FEP surface (p < 0.0001). This effect was also seen with citrated blood flowing through Mab-treated polyurethane tubing at a shear rate of 132 s(-1) (p=0.034). Since platelets and proteins (as blood, plasma or serum) were introduced to the surface simultaneously, the generation of a defined protein film must have been sufficiently rapid as to shape the platelet or cell response.     

Antibodies against GD2 ganglioside can eradicate syngeneic cancer micrometastases

After 10 years of clinical trials in patients with advanced cancer, monoclonal antibodies (mAbs) against cell surface antigens have not lived up to their initial promise. One such cell surface antigen is the ganglioside GD2. GD2 is richly expressed at the cell surfaces of human neuroblastomas, sarcomas, and melanomas. We have described a murine lymphoma (EL4) that is syngeneic in C57BL/6 mice and expresses GD2, a mAb against GD2 (mAb 3F8), and we have prepared a conjugate vaccine (GD2-keyhole limpet hemocyanin plus immunological adjuvant QS-21) that consistently induces antibodies against GD2. We demonstrate here, for the first time in a syngeneic murine model, that passively administered and vaccine-induced antiganglioside antibodies prevent outgrowth of micrometastases, and we use this model to establish some of the parameters of this protection. The level of protection was proportional to antibody titer. Treatment regimens resulting in the highest titer antibodies induced the most protection, and protection was demonstrated even when immunization was initiated after tumor challenge. Treatment with 3F8 1, 2, or 4 days after i.v. tumor challenge was highly protective, but waiting until 7 or 10 days after challenge resulted in minimal protection. The results were similar whether number of liver metastases or survival was used as the end point. These results suggest that unmodified mAbs or antibody-inducing vaccines against GD2 (and possibly other cancer cell surface antigens) should be used exclusively in the adjuvant setting, where circulating tumor cells and micrometastases are the primary targets.     

The isolation and characteristics of monoclonal antibodies to recombinant proteins of HIV-1 and HIV-2--the env and gag gene products

Ten hybridomas secreting monoclonal antibodies (Mab) against recombinant HIV-1 and HIV-2 antigens were produced (3 Mab anti-gag protein, 2 anti-env1, and 5 anti-env2). In the immunoblotting assay all the anti-gag Mabs reacted with HIV capsid protein p24, whereas one of them reacted also with p55 protein and with 7 other polypeptides. Another anti-gag Mab cross-reacted with the antigen of subpopulation of human peripheral blood lymphocytes. The third one interacted with the antigen of both HIV-1 and HIV-2. All the 10 Mabs interacted with natural HIV antigens and can be used for identification and differentiation of HIV-1 and HIV-2.     

Chimeric anti-ganglioside GM2 antibody with antitumor activity

Ganglioside GM2, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin, has been focused on as a target molecule for passive immunotherapy. GM2 is thought to be one of the T-cell-independent antigens and to elicit only IgM antibody responses in rodents and humans. We have previously established two murine anti-GM2 monoclonal antibodies with high specificity and strong binding activity, KM696 and KM697, both of which are of the IgM class. Variable heavy and light chain complementary DNAs of these two murine monoclonal antibodies were cloned and used in the construction of mouse/human IgG1 chimeric antibodies, KM966 and KM967, respectively, in this study. One of the chimeric antibodies, KM966, retained strong and specific reactivity with GM2 and showed the similarity of the binding activity with tumor cell lines to that of the original murine monoclonal antibody. Indirect immunofluorescence staining of tumor cell lines with the chimeric KM966 revealed that the antigen was expressed in substantial amounts on pulmonary tumor cells and leukemia cells as well as neuroectodermal origin tumor cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, respectively, chimeric KM966 was fully effective in killing GM2-expressing tumor cells. In addition, i.v. injection of chimeric KM966 markedly suppressed the establishment of human tumor xenografts in nude mice. Taken together, chimeric KM966 is the first antibody of the human IgG class to ganglioside GM2 and has strong antitumor activity both in vitro and in vivo. It is likely that chimeric KM966 will be a useful agent for passive immunotherapy of human cancer.     

Kinetic constants of alpha-methyl-D-glucoside transport in the chick small intestine during perinatal development

BACKGROUND: Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gp120 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-1 MN strain. OBJECTIVES: To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro. STUDY DESIGN: A set of 12-mer alanine substituted peptides were employed for epitope mapping using two ELISA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 10F10 were generated and their neutralizing capacity against the MN isolated was assessed. RESULTS: We first restricted the minimal length of the epitopes recognized by 2C4 and 10F10 to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 10F10 were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 10F10 by 50%. Finally, Fab fragments of Mab 10F10 were still able to neutralize the HIV-1 MN strain in vitro. CONCLUSION: This subtle distinction in the fine mapping of the epitope recognized by Mabs 2C4 and 10F10 should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 10F10 retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.     

Determination of the immunoglobulin class of complement-dependent cytotoxic antibodies in serum of D23 hepatoma-bearing rats

The immunoglobulin class of the complement-dependent cytotoxic antibodies in serum from D23 hepatoma-bearing rats (D23 TBS) for D23 hepatoma cells was analysed. When studied by affinity chromatography with concanavalin A, protamine, or staphylococcal protein A conjugated to Sepharose, the cytotoxic activity bound to the former two but not protein A. The binding fractions were further characterized by column chromatography on Sepharose CL-4B. The cytotoxic activity was recovered exclusively in the high molecular weight fractions corresponding to human IgM. Monitoring with IgG- or IgM-specific rabbit antibodies indicated that these high molecular weight cytotoxic fractions contained both IgG and IgM. However, fractionation of D23 TBS at low pH suggested that cytotoxicity was due to IgM antibodies rather than to immune-complexed IgG antibodies. This was supported by the findings that rabbit antirat IgM antibodies inhibited the cytotoxicity of TBS completely when added at high dilutions.     

Xenobiotic metabolizing enzymes in fish: diversity, regulation and biomarkers for pollutant exposure

The specificity, detection limit, and stability of twelve anti-Salmonella monoclonal antibodies (Mabs) were evaluated by cloth-based enzyme immunoassay (CEIA) and polymyxin-cloth based enzyme immunoassay (p-CEIA). Using the p-CEIA, five Mabs were found to react with cholate extracted lipopolysaccharide (LPS) antigens of all 44 Salmonella strains representing 19 different serogroups examined, with the exception of the one strain of serogroup-O tested. These five Mabs did not react with cholate extracts of any of 16 Gram-positive or Gram-negative non-Salmonella bacteria tested. The detection limit of purified S. typhimurium LPS antigen in the p-CEIA was approximately 40 ng for four of the Mabs and approximately 20 ng for the other Mab. Four of the five Mabs were stable during storage at 22 degrees C-23 degrees C for 24 h. These four Mabs are potentially useful for the immunodetection of Salmonella in foods and other samples.     

Human complement component C1q inhibits the infectivity of cell-free HTLV-I

Human T cell leukemia virus type I (HTLV-I) is a retrovirus that is not lysed by human serum or complement. It has not been determined, however, whether HTLV-I directly binds to complement components or whether it retains infectivity after incubation with human serum. We investigated the effects of human serum on the infectivity of cell-free HTLV-I produced by human and animal cells. Plating of vesicular stomatitis virus (HTLV-I) pseudotypes prepared in cat or human cells and formation of HTLV-I DNA after infection of cell-free HTLV-I produced by cat or human cells were markedly inhibited by treatment with fresh human serum, but not by heat-inactivated serum. HTLV-I infection was also inhibited by treatment with C2-, C3-, C6-, or C9-deficient serum, but not by C1q-deficient serum. Inhibitory activities of normal human serum against HTLV-I were neutralized by anti-C1q serum. Furthermore, purified C1q inhibited HTLV-I infection. The direct binding of C1q to HTLV-I was confirmed by comigration of C1q with HTLV-I virion upon sucrose density gradient ultracentrifugation of HTLV-I virion treated with C1q. Binding assay using synthetic envelope peptides indicated that C1q bound to an extramembrane region of the gp21 transmembrane protein. These findings indicate that the human complement component C1q inactivates HTLV-I infectivity.     

Analysis by a plaque assay of IgG- or IgM- dependent cytolytic lymphocytes in human blood

When monolayers of bovine erythrocytes (Eb) were exposed to purified human blood lymphocytes and either IgG or IgM fractions of rabbit anti-Eb serum, clear zones (plaques) appeared when Eb had been lysed by antibody-dependent effector cells (K cells). IgG-dependent plaque formation was complete by 20 h of incubation, while the IgM-dependent reaction required 40 h. The estimated minimal numbers of plaque forming cells (PFC) were 5.6% (IgG) and 2.0% (IgM) of the added lymphocytes. Inhibition experiments with human IgG or IgM indicated that different immunoglobulin receptors on the effector cells were involved in the two systems. In the IgG system, approximately 50% of the PFC had complement receptors and approximately 30% receptors for Helix pomatia A hemagglutinin (HP). In the IgM system, less than 10% of the PFC had complement receptors, while approximately 60% had HP receptors. The results suggest that a subset of human T cells had IgM-dependent K-cell potential. These cells are different from the majority of the IgG-dependent K cells.     

The role of complement and gp120-specific antibodies in virus lysis and CD4+ T cell depletion in HIV-1-infected patients

The substantial virus lysis was induced by HIV-1-infected patient serum and normal human complement serum in the presence of purified patient IgG. Non-infected CD4+ T cells coated with the whole virus or with a recombinant HIV-1 envelope gp120 and sensitised with patient IgG were also shown to be susceptible to complement-dependent lysis. The serum level of complement regulatory protein in a fluid phase, the C1-esterase inhibitor, was significantly correlated with serum concentration of C1q-circulating immune complexes (P=0.0062), but inversely with CD4+ T cell count (P < 0.0001). Accordingly, the disease progression in HIV-1-infected patients was significantly correlated with the level of complement activation as determined by serum level of C1-esterase inhibitor (P=0.0001), and inversely correlated with CD4+ cell count (P < 0. 0001) and gp120-specific antibody titre (P=0.0086). These results strongly suggest that the complement activation by gp120-specific antibodies play a very important role in virus clearance, but also in depletion of infected as well as gp120-coated non-infected CD4+ bystander T cells during the course of HIV-1 infection.     

A pilot clinical trial of two murine monoclonal antibodies fixing human complement in patients with chronic lymphatic leukaemia

The use of monoclonal antibodies (MABs) for the therapy of malignant diseases offers the potential advantage of greater target cell specificity, and therefore less toxicity. A major limitation of this therapeutic approach has been the inability of most MABs to kill the cell once bound to the target antigen. We have previously reported the development of two murine IgM MABs, WM63 (CD48) and WM66 (unclustered), that react with panleucocyte antigens widely expressed on cells from lymphoproliferative disorders, and are lytic with human complement. These antibodies have subsequently been administered intravenously to patients with chronic lymphocytic leukaemia (CLL) in a Phase One trial. Seven patients with progressive CLL received increasing daily doses of WM66 (Patients 1-3) or WM63 (Patients 4-7), with one patient also receiving a continuous infusion of WM63 over 20 hours. All patients demonstrated a significant but transient reduction in the number of circulating leucocytes, and no overall effect on disease progression was observed. Antibody coating of circulating lymphocytes was seen in patients receiving WM-63. Patients receiving large doses of WM63 (cases 5-7) demonstrated a decline in complement levels during treatment. There were no major adverse reactions to WM66, but two patients developed dose limiting side effects to WM63. No human anti-mouse antibody (HAMA) responses were documented. These findings indicate that in vitro cytotoxicity mediated by Mabs fixing human complement correlates poorly with clinical responses, and support earlier observations which indicate that cell-mediated cytotoxicity is necessary for effective antibody therapy.     

Heat shock cognate protein 71-associated peptides function as an epitope for Toxoplasma gondii-specific CD4+ CTL

HLA-DR-restricted CD4+ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4+ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4+ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4+ CTL.     

A multiaperture collimator for use at 511 keV

The addition of autologous serum to mixtures containing human red cells, from pregnant and nonpregnant females, and sheep cells resulted in the formation of mixed aggregates containing both human and sheep red cells. In contrast, no aggregate formation occurred when autologous cord serum was added to mixtures containing cord red cells and sheep red cells. Heat inactivation of the adult serum or the presence of 0.15 M EDTA prevented the formation of mixed aggregates. These observations indicated that the mixed aggregates occurred through the complement-dependent red cell immune adherence (RCIA) phenomenon. The addition of untreated cord serum to mixtures containing inactivated adult serum restored the formation of mixed aggregates, indicating that the cord serum contained sufficient complement for RCIA. Natural antibody against sheep red cells was present in adult sera but was absent in cord sera. Using the RCIA receptor assay, the RCIA receptor activity of cord red cells was found to exceed significantly that of the adult pregnant cells (p less than 0.0025). It is postulated that this may represent an aspect of immune adaptation between mother and fetus.     

Development of male contraceptive vaccine--a perspective

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.     

Complement opsonization is required for presentation of immune complexes by resting peripheral blood B cells

Complement receptor 2 (CD21, CR2) is a B cell receptor for complement degradation products bound to Ag or immune complexes. The role of CD21 in mediating Ag presentation of soluble immune complexes by resting B cells was studied. Complement-coated immune complexes were formed by the incubation of influenza virus with serum from immune donors. These complexes bound to peripheral blood B cells in a complement-dependent manner. The binding required CD21 or, to a lesser extent, complement receptor 1 (CR1, CD35). B cells pulsed with immune complexes containing complement elicited a response from a panel of influenza-specific T cell clones, while those pulsed with immune complexes formed in the absence of complement did not. The expression of the early activation marker CD69 and the costimulatory molecule CD86 were not induced by CD21 ligation alone, suggesting that CD21-mediated Ag presentation occurs independently of B cell activation. Up-regulation of these markers required exposure to T cell factors elicited by the recognition of Ag derived from complement-containing immune complexes. These findings suggest that binding of Ag to CD21 enables Ag-nonspecific B cells to participate in the activation of Ag-specific T cells in a process that occurs independently of well-characterized B cell activation events.     

D2 dopamine receptor antisense increases the activity and mRNA of tyrosine hydroxylase and aromatic L-amino acid decarboxylase in mouse brain

OBJECTIVE: Cytolytic activity of TNF was analysed at L929 and K562 tumor cell lines. METHODS: TNF-mediated cytotoxicity was studied within 10(-6)-10(-17) M concentration range after 18 h of incubation with target cells. RESULTS: TNF caused reliable cytotoxicity values in both cell lines, while L929 cells were more sensitive to cytolytic action of the protein than K562 cells. Three cytotoxicity maxima were detected at each cell line: at concentrations of 10(-6) M, 10(-17) M and 10(-15) M in K562 cells and at concentrations of 10(-7) M, 10(-11) M, 10(-14), 10(-16) M in L929 cells. CONCLUSIONS: DNA fragmentation analysis demonstrated that all cytolytic processes induced by TNF in L929 cells are associated with apoptotic mechanism of cell death, while cytolytic process induced in K562 cells differed in DNA fragmentation patterns: cytolytic processes induced by 10(-6) M of TNF was of apoptotic type, while the other processes were not associated with internucleosomal DNA cleavage.     

Sensitivity of Entamoeba histolytica and Entamoeba dispar patient isolates to human complement

Twenty-one Entamoeba histolytica and 56 Entamoeba dispar patient isolates were investigated for their sensitivity to the classical and alternative pathway of human complement, E. histolytica and E. dispar patient isolates were differentiated by polymerase chain reaction and hexokinase isoenzyme typing. It was found that 90.3% (+/- 12.0%) of the trophozoites of E. histolytica were lysed after 30 min by the alternative pathway of complement in the presence of 50% human serum (19 isolates showed lysis rates higher than 80%), whereas E. dispar cells were less susceptible to the alternative pathway as 68.8% (+/- 28.2%) of lysis occurred. However, 23 of the E. dispar isolates were lysed between 100 and 80% (90.9% +/- 9.1%), demonstrating that about half of the tested E. dispar isolates were highly sensitive to complement lysis. Only 11 of the E. dispar isolates were proven to be ?resistant' to the alternative pathway of complement and were lysed less than 40%. These results are in conflict to earlier publications, describing resistance of E. dispar to complement lysis (Hamelmann et al. 1992, 1993).     

The development of monoclonal antibodies for the therapy of cancer

Monoclonal antibodies (Mabs) were first described by K?hler and Milstein in 1975. Not only did this discovery lead to a Nobel prize, but it created an enormous scientific field that has now become a multimillion dollar industry. Mabs made the transition from laboratory reagents to clinical diagnostics very quickly. However, their development as therapeutic agents was, as predicted, more costly and time-consuming. Indeed, clinicians and scientists were required to learn a new set of rules for using these large, immunogenic, targeted agents in humans. Nevertheless, in 1997 the first Mab was licensed in the U.S. and several others will soon follow. In this review, we discuss Mab-based strategies for the treatment of cancer. We compare native, fragmented, recombinant and chimeric antibodies, bispecific antibodies, immunoconjugates, and immunoliposomes. The rationale for their development, their advantages, their in vitro and in vivo performance, and their clinical usefulness are discussed.     

In vitro development and preservation of specific features of collecting duct epithelial cells from embryonic rabbit kidney are regulated by the electrolyte environment

During kidney development the embryonic collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into functional principal (P) and intercalated (IC) cells. Aldosterone is able to modulate this differentiation. Consequently we investigated whether increased concentrations of extracellular NaCl or Na gluconate may also have an influence on the development of individual CD cell features. Embryonic CD epithelia were isolated from neonatal rabbit kidneys, placed on tissue carriers and cultured in gradient containers, which were constantly perfused with medium for 13 days. Isotonic culture conditions could be mimicked, when on both the luminal and basal side standard Iscove's Modified Dulbecco's Medium (IMDM) was used. In another set of experiments, gradient culture was performed. Standard IMDM was applied on the basal side and IMDM supplemented with 12 mM NaCl and 17 mM Na gluconate on the luminal side. This adaptation of IMDM led to the same Na concentrations as found in the serum of neonatal rabbits. The development of CD cell features was monitored by cellular markers such as the monoclonal antibodies (Mabs) 703 and 503 recognizing P and IC cell features respectively. Epithelia cultured under isotonic conditions showed less than 5% Mab 703- and 503-immunopositive cells. In contrast, epithelia cultured in a luminal-basal medium gradient revealed more than 80% positive cells. Immunoreactivity started to develop after a long lag period of 4 days, then increased continuously during the following 5 days and reached a maximum at day 14. When the medium gradient was then changed to an isotonic environment for another 5 days immunoreactivity for Mab 703 remained stable, while the number of Mab 503-positive cells was found to be decreased to 10%. Thus, the extra-cellular electrolyte environment not only induces but also preserves individual cell features.