Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells.

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca2+]i) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca2+]i. We observed small, brief, Ca2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd3+) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells.

HLA allele frequency and clinical outcome in Italian patients with cutaneous melanoma.

The current study focuses the analysis on the possible relationship between HLA allele frequency and clinical outcome of melanoma in a population of 382 Italian patients, as compared with 203 ethnically matched controls. In a 3-year follow-up study, results showed significant differences between groups of patients selected according to clinical stage, histology, and progression of the disease. A*01 seems to be correlated with a less aggressive variant of the disease, whereas DRB1*01-DQB1*0501 seems to be associated with metastatic progression of melanoma. Moreover, a negative association with B*13, B*44, as well as with DRB1*04-DQB1*0302 was found. A multivariate logistic regression model showed HLA-DRB1*04 to behave as an independent favorable prognostic marker of melanoma in our population (OR = 2.34, CI = 1.15-4.74).

Myelin basic protein gene is associated with MS in DR4- and DR5-positive Italians and Russians.

BACKGROUND: The myelin basic protein (MBP) gene may confer genetic susceptibility to multiple sclerosis (MS). The association of MS with alleles of the (TGGA)n variable number tandem repeat (VNTR) 5' to the MBP gene is the subject of conflicting reports. OBJECTIVE: To study possible MS association with VNTR alleles of MBP gene in ethnic Italians and ethnic Russians. METHODS: Two hundred sixty-nine unrelated patients with definite MS and 385 unrelated healthy control subjects from Italy and Russia were genotyped for the MBP VNTR region and for the human leukocyte antigen (HLA) class II DRB1 gene. The phenotype, allele, and genotype frequencies for two groups of MBP alleles were determined. Patients and control subjects were stratified according to HLA-DRB1 phenotypes. RESULTS: The distribution of MBP alleles and genotypes in the two ethnic groups, including both MS patients and control subjects, was very similar. When MS patients and healthy control subjects were stratified according to HLA-DRB1 phenotypes, a significant association of MS with MBP alleles was found only in the DR4- and DR5-positive subgroups. A significant association with MBP alleles was also observed in the nonstratified groups, owing mainly to the contribution of the DR4- and DR5-positive individuals. CONCLUSION: Polymorphism of the MBP or another gene in its vicinity appears to contribute to the etiology of MS for the subgroups of DR4- and DR5-positive Italians and Russians.

HLA-DRB1*1501 and response to copolymer-1 therapy in relapsing-remitting multiple sclerosis.

BACKGROUND: Copolymer 1 (Cop-1) is a random synthetic amino acid copolymer, effective in the treatment of the relapsing-remitting form of MS (RRMS). In vitro and in vivo studies suggest that the mechanism of Cop-1 involves its binding to major histocompatibility complex class II molecules as an initial step. OBJECTIVE: To assess a possible relationship between human leukocyte antigen (HLA) alleles and response to Cop-1 therapy. METHODS: Eighty-three patients with RRMS, 44 treated with Cop-1 and 39 with interferon beta-1a (IFNbeta-1a) for 2 years, were typed by molecular methods for HLA class II genes and subgrouped according to clinical outcome. RESULTS: Data have shown a possible positive correlation between presence of DRB1*1501 and response to Cop-1 therapy (p = 0.008). No relationship between HLA alleles and therapy has been found in IFNbeta-1a treated patients. CONCLUSIONS: Results suggest that DRB1*1501 might be relevant for the clinical outcome in Cop-1 treated patients and, if confirmed in larger studies, it could be helpful in the selection of RRMS patients for different therapeutic options.

Crohn disease: susceptibility and disease heterogeneity revealed by HLA genotyping.

Predisposition to Crohn disease (CD) seems to be genetically determined but, though several reports on the matter, the association between HLA antigens and the disease is still controversial. PCR-SSP high resolution typing in 107 CD patients, and in subgroups selected according to clinical features, showed a positive association with the rare haplotype DRB1*07, DQB1*0303 both in the overall patients (p = 0.002; pc = ns) and in the subgroup of nonfistulized patients (p = 0.0008; pc = 0.032). Moreover, the protective role of the haplotype DRB1*03, DQB1*0201 (p = 0.029) was confirmed also in Italian patients, whereas no strong association with HLA class I alleles has been found. In addition, variability of the HLA alleles frequency in CD subgroups was observed, supporting the hypothesis of a genetic heterogeneity of the disease and suggesting that HLA alleles distribution in selected groups may allow to identify patients with probably different prognosis or associated complications.

In vitro tannin acantholysis.

BACKGROUND: Exogenous factors, such as certain drugs, may be involved in the induction of pemphigus. Other offenders sharing a similar chemical composition to these drugs may also play a role. Tannins with their considerable biologic activity were suggested as possible factors. To substantiate the role of tannins in the pathomechanism of pemphigus, the present study examined the acantholytic potential of tannins in vitro. METHODS: Normal human breast skin from patients without any bullous disease was cultured for 3 days in the presence of tannic acid at concentrations of 0.02, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 mM. The effect of the tannic acid was microscopically examined in a blind fashion by three independent investigators. RESULTS: In addition to the cytotoxic effect, tannic acid caused marked acantholytic changes, with a clear suprabasal cleavage and intraepidermal acantholytic cells. The acantholytic changes were the most constant and specific effects. They were constantly observed at 1.0 and 2.0 mM, whereas lower concentrations showed changes only in some of the explants. The concentrations needed to exert this effect were notably low. There was a remarkable variability among the subjects who had provided the explants. CONCLUSIONS: The results suggest a possible role of tannin in the disease process of pemphigus. The tannin acantholytic potential was much greater than the potential of known acantholytic drugs, such as penicillamine and captopril. The interindividual variability in susceptibility to acantholysis may explain the variability in the individual potential for developing pemphigus.

Effects of lithium carbonate (Li2CO3) on in-vitro-cultured normal human skin explants.

The early morphological changes induced by lithium carbonate, a well-known psoriasis-provoking drug, were studied on cultured skin. Normal human skin from patients undergoing mastectomy was cultured in the presence of 3 mM, 6 mM and 10 mM of Li2CO3 for 4 days. The morphological changes were then evaluated by three observers in a blind manner and their reports were matched and collated. The cultured skin in the presence of Li2CO3 showed cell crowding of keratinocytes in the lower part of the epidermis, indicating epidermal hyperplasia. Another striking finding was intercellular oedema and vacuolar alteration with formation of small cavities in the upper dermis. There was no evidence of parakeratosis or any other histological characteristic of psoriasis, except hyperproliferation of the epidermis. Based on our knowledge of mechanisms of lithium action, we proposed two competitive explanations for its action on the epidermis: i) that lithium acts directly on dividing cells of the epidermis; and ii) that it acts indirectly by altering epidermal barrier function. Although we lack definite proof, we suggest that the observed morphological changes, in particular the non-specific stimulus to epidermal proliferation, are the primary events which initiate the process that will ultimately lead to the development of psoriasis in a predisposed patient.

Implication of tissue transglutaminase and desmoplakin in cell adhesion mechanism in human epidermis.

The distribution patterns of both tissue and keratinocyte transglutaminases (TGase), as well as that of desmoplakin (DP), have been immunohistochemically investigated in human skin cultured in the absence or presence of cystamine and enalapril, two acantholytic agents. In the control samples, tissue TGase is predominantly expressed in lower layers of the epidermis and is located intercellularly. Conversely, in tissues cultured with cystamine or enalapril, a diffuse cytoplasmatic staining was observed. Similarly, DP, detected on the cell membrane in the control, shifts into the cytosol of the keratinocytes following treatment. The distribution pattern of the keratinocyte enzyme in the acantholytic epidermis was identical to that observed in the normal one. Since cystamine and enalapril are TGase inhibitors and DP was shown to act as a TGase substrate in vitro, we suggest that DP and tissue enzyme may participate in cell adhesion at the intraepidermal level.

CD40 expressed on human melanoma cells mediates T cell co-stimulation and tumor cell growth.

CD40 is a 50 kDa molecule, a member of the tumor necrosis factor/nerve growth factor receptor family. It is expressed on B cells, monocytes, dendritic cells and various malignant cells. While the critical relevance of this molecule in T cell-dependent B cell activation is already established, the biological role of CD40-CD154 interaction in non-hematopoietic cells is still unknown. Here we show that CD40 is functionally expressed on human melanoma-derived cell lines. No correlation between surface CD40 expression and the origin of the cell line, primary versus metastatic, was observed. Melanoma cells were shown to be able to co-stimulate TCR-triggered human T cells; moreover, because they do not express CD80 or CD86 co-stimulatory structures, the involvement of additional pathways have to be postulated. We have identified CD40 as one of the molecules involved in melanoma cell-mediated co-stimulation of anti-CD3-triggered human CD4(+) T lymphocytes. In addition, a CD40-dependent pathway, able to enhance tumor cell proliferation at low serum concentrations, in vitro, has been shown to be functional in human melanoma cell lines.

Increased serum concentrations of soluble HLA-class I antigens in hepatitis C virus related mixed cryoglobulinaemia.

OBJECTIVE: To investigate whether quantitative alterations of both beta(2)microglobulin (beta(2)micro) associated HLA class I heavy chains (sHLA-I) and beta(2) micro free class I heavy chains (sHLA-FHC) in sera of patients with hepatitis C virus (HCV) infection occur and whether they distinguish patients with mixed cryoglobulinaemia (MC). METHODS: 83 HCV infected patients were studied and divided into three groups: (A) without cryoglobulinaemia (n=21), (B) with polyclonal MC (n=20), (C) with monoclonal MC (n=42). Serum sHLA-I and sHLA-FHC were measured by double determinant radioimmunoassay using monoclonal antibodies: TP25.99 as catching antibody, and NAMB-1 and HC-10 as revealing antibodies. Western blot identified HLA-I isoforms. RESULTS: The serum concentrations of sHLA-I and of sHLA-FHC in HCV infected patients versus controls were respectively 1.3(0.5) microg/ml (mean (SD)) versus 0.8 (0.3) (p<0. 001) and 13.9 (7.1) ng/ml versus 9.2 (5) (p<0.001). sHLA-I were 1.01 (0.4) microg/ml in group A, 1.04 (0.4) microg/ml in group B, and 1. 47 (0.4) microg/ml in group C (p=0.001). Statistical analysis showed a significant difference versus controls for groups B (p<0.02) and C (p<0.001). sHLA-FHC were 12.8 (8.3) ng/ml in group A, 17.2 (7.1) ng/ml in group B, and 12.9 (6.2) ng/ml in group C (p<0.02). A significant difference versus controls for each group was found (p<0. 02, p<0.001, and p<0.02, respectively). Different patterns of sHLA-I isoforms were observed. CONCLUSIONS: Increased serum concentrations of sHLA-I and sHLA-FHC characterise HCV infected patients. The highest sHLA-I concentrations seem to distinguish patients with monoclonal MC. In this last condition sHLA could play a part in the HCV escape and in B cell proliferation. The significance of sHLA-FHC is still undefined.

Detection of herpesvirus DNA in peripheral blood mononuclear cells and skin lesions of patients with pemphigus by polymerase chain reaction.

Pemphigus is an autoimmune disease where both endogenous (genetic) and exogenous (environmental) factors play a part. Viral infections, in particular herpesvirus infections, have been identified as a possible triggering factor for pemphigus. In this study, using the polymerase chain reaction, we studied peripheral blood mononuclear cells (PBMC) and skin biopsies from patients with pemphigus, and in some of these were able to demonstrate the presence of DNA sequences of herpes simplex virus 1/2 (50% in PBMC and 71% in skin biopsies), Epstein-Barr virus (15% in PBMC and 5% in skin biopsies) and human herpesvirus 6 (20% in PBMC only). However, the inability to detect herpesvirus DNA consistently in these cases suggests that viral infection may only be an occasional factor triggering the outbreak or exacerbation of the disease. The possible role of interferons and interleukins in the pathogenesis of virus-induced pemphigus is discussed.

Common human leukocyte antigen alleles in pemphigus vulgaris and pemphigus foliaceus Italian patients.

Pemphigus refers to a group of autoimmune blistering skin diseases, mainly identified as pemphigus vulgaris and pemphigus foliaceus, both characterized by the presence of autoantibodies against keratinocyte adhesion molecules, leading to loss of cell-cell adhesion with consequent blister formation. Pemphigus vulgaris is reported to be associated with human leukocyte antigen DR4 and/or DR6 whereas no data are available on pemphigus foliaceus, except for the endemic Brazilian form (fogo selvagem), which is reported to be associated with DR1 and DR4. We here report human leukocyte antigen molecular typing on a total of 87 patients, 61 with pemphigus vulgaris and 26 with pemphigus foliaceus, versus 128 healthy matched controls. Generic typing showed an increase of DRB1*04 and DRB1*14 and a decrease of DRB1*07 in both pemphigus vulgaris and pemphigus foliaceus patients. Molecular subtyping of DR4+ and DR14+ subjects showed a highly significant association between the DRB1*1401 and both pemphigus vulgaris (p < 0.0001) and pemphigus foliaceus patients (p < 0.0001) together with a significant increase of the linked DQB1*0503 (pemphigus vulgaris p < 0.0001; pemphigus foliaceus p < 0.0001). Moreover, whereas the association between DRB1*0402 and pemphigus vulgaris (p < 0.0001) has been confirmed, no significant association between a specific allele of the DR4 group and pemphigus foliaceus, has been found. Therefore, at least in Italian patients, pemphigus vulgaris and pemphigus foliaceus share DRB1*1401 and DQB1*0503, as susceptible human leukocyte antigen alleles, whereas DRB1*0402 is only found associated with pemphigus vulgaris. The observation that both diseases, pemphigus vulgaris and pemphigus foliaceus, carry the same susceptible human leukocyte antigen alleles has been interpreted as a common genetic background predisposing to pemphigus as, like in other autoimmune disorders, it is not sufficient to explain the onset of the disease on the basis of the sole aforementioned alleles. Other linked genes and/or environmental factors should play a facilitating role in the outbreak of pemphigus, either pemphigus vulgaris or pemphigus foliaceus.

The in vitro effect of hydroxychloroquine on skin morphology in psoriasis.

BACKGROUND: In earlier papers, we suggested that the aggravation of psoriasis by antimalarial drugs (analogous to hypolipidemic drugs) could be initiated by a break in the epidermal barrier. We suggested that these drugs exerted their effect by inhibiting epidermal transglutaminase activity, and supported this hypothesis by demonstrating the effect of hydroxychloroquine sulfate (HCQS) on the morphology of cultured skin and on liver transglutaminase activity. In the present article, we describe, for the first time, the morphologic changes induced by HCQS on cultured skin of psoriatic patients. METHODS: Uninvolved (apparently normal) skin explanted from the back of two psoriatic patients was cultured in the presence of 9.2 and 13.8 mM of HCQS for 3 days. The morphologic changes were evaluated in a blind manner. The experiment was repeated twice. RESULTS: Significant changes in the epidermal morphology of psoriatic skin cultured in the presence of HCQS, compared with skin cultured without the presence of the drug, were observed. The most striking changes were enhanced and irregular keratinization and dermo-epidermal detachment and cleft formation. No parakeratosis or other characteristics of psoriasis were observed. CONCLUSIONS: The first changes caused by HCQS on the cultured skin of psoriatic patients are not characteristic of psoriasis, and include hyperproliferation and enhanced and irregular keratinization. The present experimental study gives further support to the hypothesis that HCQS causes an initial break in the barrier function of the epidermis (probably by inhibiting transglutaminase activity), which is followed by a physiologic response of the epidermis aimed at barrier restoration. This rather nonspecific stimulus to epidermal proliferation is probably sufficient to trigger psoriasis, in vivo, among genetically predisposed patients.

Pemphigus and pemphigoid-like effects of nifedipine on in vitro cultured normal human skin explants.

BACKGROUND: A variety of drugs have been implicated in the onset and exacerbation of pemphigus and bullous pemphigoid. The demonstration of biochemical acantholysis in skin explants to various drugs in the absence of autoantibodies, in which the tested drugs evoke a biochemical reaction that leads to desmosomal function loss, may be a valuable adjunct to patient management by confirming the suspicion of drug-related pemphigus or bullous pemphigoid. OBJECTIVE: To determine whether a skin explant model might serve as a possible in vitro correlate of drug-induced pemphigus and pemphigoid-like effects related to the calcium channel blocker nifedipine. METHODS: Normal human breast skin obtained from nonpemphigus and nonpemphigoid patients undergoing mastectomy was cultured with nifedipine at final concentrations of 2, 4, and 8 mM. The drug effect on skin explants evidenced by morphologic changes was evaluated by microscopy by three observers. RESULTS: Five out of seven explants cultured with nifedipine at concentrations ranging from 2 to 8 mM exhibited obvious morphologic changes of two types: intraepithelial (or pemphigus-type) splittings and subepithelial (or pemphigoid-type) splittings. Two explants showed no acantholysis and no subepithelial splittings. Control cultures without polyethylene glycol 200 (PEG) showed no changes. Skin control samples cultured in medium supplemented with 10% PEG displayed vacuolar degeneration throughout the entire epidermis, but no sign of cell-cell dyshesion or dermo-epidermal detachment. CONCLUSIONS: A type of skin susceptibility to nifedipine may be genetically determined, with some nifedipine-treated patients developing an acantholytic reaction and others a subepidermal bullous eruption.

Molecular analysis of HLA DRB1 and DQB1 polymorphism in Italian melanoma patients.

Several studies have reported association between a variety of malignancies and human leukocyte antigens (HLA) genes. However, conflicting data have been reported on HLA association and melanoma. We report here serologic and molecular analysis by polymerase chain reaction sequence-specific primers (PCR-SSP) and PCR sequence-specific oligonucleotides (PCR-SSO) of HLA class II DRB1 and DQB1 loci in 132 patients with melanoma and 102 ethnically matched controls. Molecular typing of DQB1 polymorphism showed a significant increase of DQB1 *0501 (25.0% versus 14.7%; p = 0.038). Moreover, an increase of DQB1*0301, which was present in 62.8% of patients and 54.9% of controls (p = 0.136), was noted. Because DQB1*0501 and DQB1*0301 are strongly linked to DRB1*01 and DRB1*11, respectively, both found increased in patients with melanoma, to look for a more stringent association with a particular allele specificity of the DR locus, we performed PCR-SSP high-resolution typing of DR1 and DR11 positive subjects. Results showed no significant difference between the frequencies of the alleles found in patients with respect to controls. Analysis of the distribution of DQB1*0501 and DQB1*0301 according to the AJCC clinical stage of the disease showed no significant difference in the frequency of these alleles between the localized and the metastatic form of the disease. However, none of the HLA class II alleles showed significant association after correction of the p value. These results indicate that HLA class II alleles may not contribute to a strong susceptibility to melanoma, at least in Italian patients.

Increased level of serum HLA class I antigens in patients with systemic lupus in patients with systemic lupus erythematosus. Correlation with disease activity.

The level of soluble beta2-mu-associated HLA Class I heavy chains (sHLA-I) and of soluble beta2-mu-free HLA Class I heavy chains (sHLA-FHC) was found to be significantly higher in sera from 58 patients with systemic lupus erythematosus (SLE) than in those from 82 age and sex-matched controls. The level of serum sHLA-I in patients with SLE was significantly correlated to disease activity. Western blotting analysis showed that the 44-kDa isoform represents the major component in the antigens immunoprecipitated by anti-beta2-mu mAb NAMB-1 and by anti-beta2-mu-free HLA Class I heavy chain mAb HC-10 from sera of patients with SLE. These results suggest that the increased serum levels of sHLA-I and of sHLA-FHC in patients with SLE reflect their increased shedding from cell membrane. In view of the ability of sHLA-I and of sHLA-FHC to induce apoptosis of activated T cells, it is suggested that their increased serum levels in patients with SLE is triggered by dysregulation of the immune system leading to T-cell activation. The increased serum levels of sHLA-I and of sHLA-FHC may be used by the immune system to control the pool of activated T cells by inducing apoptosis. If this possibility is proven to be correct, modulation of the serum level of sHLA-I and of sHLA-FHC may be utilized to develop strategies to treat SLE.

Different patterns of in vitro acantholysis in normal human skin samples explanted from different sites of the body.

BACKGROUND: The factors that contribute to a preferential anatomic localization of pemphigus lesions are not well known. In particular, the question arises as to whether certain skin areas may be more acantholysis-prone than others. OBJECTIVE: To verify whether, in pemphigus patients, a different susceptibility to acantholysis exists among different cutaneous regions, the technique of tissue cultures was used. METHODS: Normal human skin explants from two distinct anatomic regions (back and buttocks) of two former pemphigus patients were cultured in vitro in the presence of enalapril (6 mM) or cystamine (10 mM), two substances with a proven biochemical acantholytic effect. After 4 days of culture, the tissues were processed for standard histology. RESULTS: Diffuse acantholysis, with large intraepidermal splits, was observed in the explants taken from the backs of both subjects and cultured with either enalapril or cystamine. Mild to moderate acantholytic changes were detected in the explants taken from the buttocks of both subjects and cultured with either enalapril or cystamine. No structural changes were seen in the control cultures. CONCLUSIONS: Pemphigus patients present different thresholds of acantholysis in different areas of their bodies. This might explain, at least in part, certain preferential anatomic localizations of pemphigus lesions.

The in vitro effect of hydroxychloroquine on skin morphology and transglutaminase.

BACKGROUND: Antimalarials are some of the most notorious drugs which may induce psoriasis, with 25% of all reported cases being associated with them. Antimalarials do not induce psoriasis de novo, but trigger subclinical psoriasis. In a previous report, we suggested that antimalarials exert their effect by interfering with the epidermal transglutaminase (TGase) activity. OBJECTIVE: To verify this hypothesis by examining the effect of hydroxychloroquine sulfate (HCQS) on cultured human skin and on TGase activity in vitro. MATERIALS AND METHODS: Skin samples from normal donors were cultured in the presence of HCQS for 4 days, and then processed for microscopic examination. TGase activity was assayed in the presence of HCQS and compared with blanks. RESULTS: Significant changes in epidermal morphology were seen in all explants cultured in the presence of HCQS at all concentrations employed. Areas of enhanced and irregular keratinization were observed in the upper epidermis, while a loss of cell polarity, with keratinocyte crowding and disarray, was seen in the lower epidermis. In addition, we observed intraepidermal splitting at different levels and dermo-epidermal detachments. HCQS showed a concentration-dependent inhibition of TGase activity. CONCLUSIONS: We suggest that HCQS causes an initial break in the barrier function of the epidermis by inhibiting TGase activity; this is followed by a physiologic response of the epidermis aimed at barrier restoration. This rather nonspecific stimulus to epidermal proliferation is probably sufficient to trigger psoriasis in predisposed individuals or aggravate it in psoriatic patients.

JURL-MK1 (c-kit(high)/CD30-/CD40-) and JURL-MK2 (c-kit(low)/CD30+/CD40+) cell lines: 'two-sided' model for investigating leukemic megakaryocytopoiesis.

Two novel cell lines (JURL-MK1 and JURL-MK2) have been established from the peripheral blood of a patient in the blastic phase of chronic myelogenous leukemia. The cells grow in a single cell suspension with doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2). Cytogenetic analysis has shown that JURL-MK1 is hypodiploid whereas JURL-MK2 is near triploid and that both cell lines retain t(9;22). Moreover, JURL-MK1 and JURL-MK2 have a bcr/abl-fused gene with the same junction found in the patient's fresh cells, and both cell lines express the b3/a2 type of hybrid bcr/abl mRNA. The morphology and immunophenotype of these cell lines are reminiscent of megakaryoblasts. In both lines, a limited but consistent percentage of cells expresses gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gplb (CD42b), glycophorin A, hemoglobin and CD34. Both cell lines are clearly positive for CD33, CD43, CD45RO and CD63, while CD13, CD44, CD54, CD30 and CD40 are specific features of JURL-MK2. Among cytokine receptors, CD117/SCF-R is strongly displayed by a large fraction of JURL-MK1 cells but is hardly detectable on about 20% JURL-MK2 cells. Both cell lines are clearly positive for CD25/IL2R alpha, while a marked expression of CD116/GM-CSF-R and CDw123/IL3R alpha is restricted to JURL-MK2. Induction of cell differentiation in vitro has demonstrated that TPA is able to modulate the JURL-MK1 phenotype, causing an increased expression of platelet-associated antigens. The JURL-MK2 phenotype is easily modulated by both TPA and DMSO, which cause an increased expression of CD41a and CD117 accompanied by a decreased expression of CD30. Proliferation studies demonstrated that JURL-MK1 cell growth is enhanced by stem cell factor, while JURL-MK2 proliferation is unaffected by this cytokine. JURL-MK1 and JURL-MK2 are two novel cell lines with divergent biological features, representing a 'two-sided' model for investigating new aspects of megakaryocytopoiesis.