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Recently, a crucial role of Th2 responses in nickel allergic contact dermatitis ( ACD) was demonstrated. As palladium allergy is an issue of growing interest, the diagnostic potential of Th2 parameters for palladium sensitization was investigated. Palladium (Na2[PdCl4])-induced lymphocyte proliferation ( LPT), Th1 and Th2 cytokine production were correlated with skin test ( ST) reactivity in 16 positive and 21 negative controls. Furthermore, the diagnostic potential of these assays was evaluated using receiver operating characteristics ( ROC) analysis. For comparison, same experiments were carried out for nickel (Ni SO4). Correlation coefficients between palladium ST reactivity and IFN-γ, LPT, IL-5, and IL-13 were 0.34, 0.51, 0.69, and 0.78, and overall test accuracies were 68%, 81%, 89%, and 95%, respectively. Both palladium- and nickel-mediated Th2 responses tightly correlate with ST reactivity, supporting recent findings on the crucial role of Th2 involvement in ACD. Therefore, these assays may have great potential as diagnostic tools for future in vitro sensitization testing. [ABSTRACT FROM AUTHOR]

Tumor-derived vascular endothelial growth factor (VEGF) has previously been identified as a causative factor in the disturbed differentiation of myeloid dendritic cells (DC) in advanced cancer patients. Here, we investigated the potential of vascular endothelial growth factor receptor (VEGFR) tyrosine kinase (TK) inhibition to overcome this defective DC differentiation. To this end, peripheral blood DC (PBDC) precursor and subset frequencies were measured in 13 patients with advanced cancer before and after treatment with AZD2171, a TK inhibitor (TKI) of VEGFR, coadministered with gefitinib, and an epidermal growth factor receptor (EGFR) TKI. Of note, not only myeloid DC but also plasmacytoid DC frequencies were significantly reduced in the blood of the cancer patients prior to treatment, as compared to healthy controls. Moreover, besides an accumulated population of immaturemyeloid cells (ImC), a population ofmyeloid suppressor cells (MSC) was significantly increased. Upon systemic VEGFR TK inhibition, DC frequencies did not increase, whereas the rate of circulating MSC showed a slight, but not significant, decrease. In conclusion, TK inhibition of VEGFR with AZD2171 does not restore the defective PBDC differentiation observed in advanced cancer patients. [ABSTRACT FROM AUTHOR]

Multidrug transporters, such as P-glycoprotein (P-gp), multidrug-resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP), are associated with multidrug resistance in cancers; other molecules, such as major vault protein (MVP), have a similar association with drug-resistant cancer. These proteins are postulated to generate drug resistance in epilepsy. They have been shown individually to be up-regulated in epileptogenic brain tissue. In any consideration of the function, inhibition or evasion of the activity of such proteins, the colocalization of such proteins needs to be understood. We systematically determined the presence of such colocalization, focusing on microvascular endothelium from epileptogenic human brain tissue. Double labelling immunofluorescence and confocal laser scanning microscopy were used to determine colocalization of P-gp, MRP1, BCRP and MVP in one case of hippocampal sclerosis and two cases of focal cortical dysplasia type IIb. Endothelial colocalization was examined with double labelling using antibodies to CD34 and Factor VIII. The presence of P-gp, BCRP and MVP in microvascular endothelium was confirmed. P-gp, BCRP and MVP colocalized in microvascular endothelium, though not all proteins appeared to be identically distributed within this tissue. MRP1 did not colocalize to endothelium. These findings were not unexpected but required formal confirmation. The demonstrated colocalization of P-gp, BCRP and MVP in microvascular endothelium in epileptogenic human brain tissue has important implications for functional experiments (including single knock-out mice studies), work with specific and broad-spectrum inhibitors of transport function, and any eventual trials of treatment of refractory epilepsy involving modulation of the function of these proteins. [ABSTRACT FROM AUTHOR]

In this study, we investigated the capacity of CD4+CD25+ regulatory T cells to suppress nickel-specific effector T cells, both in nickel-allergic patients and healthy controls. CD4+ cells isolated from allergic patients showed an increased proliferative response to nickel, whereas CD4+ cells from negative controls did not respond to allergen. When CD4+CD25+ cells were depleted, nickel-specific responsiveness was strongly increased both in allergic and in non-allergic individuals, with the most pronounced effect in allergic patients. These regulatory T cells were anergic to nickel but inhibited nickel-specific CD4+CD25– effector T cells in coculture experiments. CD4+CD25+ cells from nickel-allergic patients showed only a limited capacity to suppress effector T-cell responsiveness, because an increased nickel reactivity could still be detected in these cocultures. None of the isolated CD4+CD25+ cells, either isolated from healthy controls or allergic patients, produced IL-10 in response to nickel. Overall, these results support the view that CD4+CD25+ cells can control the activation of nickel-specific effector T cells in non-allergic individuals, whereas this regulatory capacity is impaired in allergic patients. To investigate the presence of allergen-specific regulatory T cells in truly naïve, non-sensitized individuals, T-cell reactivity should also be studied with non-environmental contact allergens, such as para-phenylenediamine. [ABSTRACT FROM AUTHOR]

Spiekstra SW, Toebak MJ, Sampat-Sardjoepersad S, van Beek PJ, Boorsma DM, Stoof TJ, von Blomberg BME, Scheper RJ, Bruynzeel DP, Rustemeyer T, Gibbs S. Induction of cytokine (interleukin-1α and tumor necrosis factor-α) and chemokine (CCL20, CCL27, and CXCL8) alarm signals after allergen and irritant exposure.The immune system is called into action by alarm signals generated from injured tissues. We examined the nature of these alarm signals after exposure of skin residential cells to contact allergens (nickel sulfate and potassium dichromate) and a contact irritant[sodium dodecyl sulfate (SDS)].Nickel sulfate, potassium dichromate, and SDS were applied topically to the stratum corneum of human skin equivalents. A similar concentration-dependent increase in chemokine (CCL20, CCL27, and CXCL8) secretion was observed for all three chemicals. Exposure to nickel sulfate and SDS was investigated in more detail: similar to chemokine secretion, no difference was observed in the time- and concentration-dependent increase in pro-inflammatory cytokine[interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α)] secretion. Maximal increase in IL-1α secretion occurred within 2 h after exposure to both nickel sulfate and SDS and prior to increased chemokine secretion. TNF-α secretion was detectable 8 h after chemical exposure. After allergen or irritant exposure, increased CCL20 and CXCL8, but not CCL27, secretion was inhibited by neutralizing human antibodies to either IL-1α or TNF-α.Our data show that alarm signals consist of primary and secondary signals. IL-1α and TNF-α are released as primary alarm signals, which trigger the release of secondary chemokine (CCL20 and CXCL8) alarm signals. However, some chemokines, for example, CCL27 can be secreted in an IL-1α and TNF-α independent manner. Our data suggest that skin residential cells respond to both allergen and irritant exposure by releasing mediators that initiate infiltration of immune responsive cells into the skin. [ABSTRACT FROM AUTHOR]

Diagnostic patch testing in allergic contact dermatitis faces the risk of boosting existing hypersensitivities or active sensitization. Risk-free and reliable in vitro assays using peripheral blood are, therefore, wanted. Here, we studied new approaches for in vitro monitoring of nickel-specific effector ad regulatory cell functions in allergic patients and potentially tolerized individuals. Lymphocyte proliferation assays were carried out with the allergen and additional IL-12/IL-7 or IL-4/IL-7 cytokine supplements. Release of IFN-γ and IL-5 were assessed as measures for type-1 and type-2 effector T cell function, respectively, and IL-10 and TGF-β1 to monitor possible regulatory T cell function reflecting immunological tolerance. After optimization of in vitro cut-off values, potency of these parameters was evaluated as compared with conventional nickel patch testing. One hundred and fifty six outpatients were included in this study, 74 of whom presenting with a positive history of nickel allergy. Nickel-sulphate patch test results showed positive reactions in 43 patients, of whom 40 had a positive history (test sensitivity 54%; specificity 96%; overall accuracy 76%). Proliferation tests without cytokine supplementation showed an accuracy of 68%, which was further improved by supplementing IL-4/IL-7 (82%). IFN-γ and IL-5 cytokine production, as revealed in IL-12/IL-7 and IL-4/IL-7 supplemented cultures, respectively, showed accuracies of 70% and 83%. As to the production of putatively immunoregulatory cytokines, IL-10 was most informative, with highest production rates in nickel-skin test negative individuals with long-lasting mucosal metal contact preceding skin piercing. These results indicate that measuring both T cell proliferation and cytokine secretion profiles, in particular IL-5 release using IL-4/IL-7 supplemented medium, offers a promising improvement of the in vitro diagnostic options in monitoring nickel contact sensitization. Since oral nickel contact has been shown earlier to induce active tolerization, nickel-induced in vitro IL-10 production may help identify nickel-tolerized individuals. [ABSTRACT FROM AUTHOR]

Abstract: Allergen-induced emigration and maturation of dendritic cells (DC) are pivotal steps in sparking off allergic contact dermatitis. In vitro models, reflecting these steps, may provide tools for assessment of sensitizing capacities of putative contact allergens. Here, we evaluated the applicability of such models for a panel of methacrylate congeners, the sensitizing properties of which were established previously in clinical and experimental animal studies. First, using interleukin-4 (IL-4)/granulocyte–macrophage colony-stimulating factor (GM-CSF)-induced, blood monocyte-derived DC, hapten-induced up-regulation of maturation/activation markers, including CD80, CD83, CD86, chemokine receptors CXCR4 and CCR5, as well as the drug resistance related molecules P -glycoprotein (Pgp) and lung resistance protein (LRP), were monitored by flow cytometry. Of note, whereas CD86 and CXCR4 were most sensitive in discriminating between the contact sensitizers and irritants included in the panel, i.e. sodium dodecyl sulphate (SDS) and croton oil (CO), assessment of CD83 and LRP expression reflected the relatively lower sensitizing capacity of methyl methacrylate. Second, using ex vivo skin explant cultures, allergen-induced LC migration from epidermal to basal membranous and dermal skin structures was most reliably monitored by CD1a, as compared with Pgp, LRP, HLA-DR or CD54 staining. The extent of CD1a[sup +] LC migration was found to closely correlate with the sensitizing capacities of the panel of test compounds. These results support the view that both in vitro models can provide valuable data on contact sensitizing properties, and add chemokine receptors and drug resistance related molecules to the list of DC membrane markers revealing allergenic signaling. [ABSTRACT FROM AUTHOR]

Summary The α‐galactosylceramide KRN7000 was reported to be presented by CD1d to natural killer (NK) T cells, cells that are thought to play an important role in the rejection of malignant tumours and in the regulation of several autoimmune diseases. Here we analysed human peripheral blood (PB) NK T cells (Vα24+ Vβ11+ T cells) before and after a short‐term culture in the presence of KRN7000. KRN7000 strongly activated PB Vα24+ Vβ11+ T cells and, when stimulated, the vast majority of these cells expressed interferon‐γ (IFN‐γ). Exposure of these KRN7000‐cultured Vα24+ Vβ11+ T cells to interleukin‐12 (IL‐12), but not to IL‐7, resulted in a relative increase in IFN‐γ‐expressing Vα24+ Vβ11+ T cells, compared with IL‐4‐expressing Vα24+ Vβ11+ T cells, indicating a shift towards a T‐helper type 1 (Th1) phenotype. KRN7000 strongly up‐regulated the expression of the cytotoxic molecule granzyme B (GrB) in Vα24+ Vβ11+ T cells. Although IL‐7 resulted in a decrease in GrB levels in KRN7000‐cultured Vα24+ Vβ11+ T cells, IL‐12 increased GrB levels in both Vα24+ Vβ11+ T cells and in Vα24+ Vβ11+ T‐cell clones and increased cytotoxicity against hCD1d‐transfected HeLa cells. Our data provide further insight into the characteristics of human Vα24+ Vβ11+ T cells and indicate that KRN7000 is a potent activator of Vα24+ Vβ11+ T cells. Combined with the established anti‐tumour effects of KRN7000 in mouse models, these results may support the use of KRN7000 as an anti‐tumour... [ABSTRACT FROM AUTHOR]

During clinical trials, a clonidine transdermal device has been found to induce clonidine-specific allergic contact dermatitis in up to 25% of patients during a treatment period of year. Using 3 different guinea pig strains, development was attempted of an experimental guinea pig model that would allow for in-depth studies into the mechanism of sensitization, and a possible role of transdermal device components. Transient low-level clonidine allergy could be obtained only in a minority of animal, with severe sensitization procedures departing from epicutaneous applications, combined with intradermal (adjuvant) FCA injections. Sensitization was not potentiated by additional booster procedures, including cyclophosphamide pretreatment, nor any of the putative cofactors (UV-treatments. C. parvum or acetaldehyde involvement) studied. These results suggest that the persistent skin contacts in man, with transdermal devices for sustained drug delivery, generate unique conditions favouring the development of allergic contact dermatitis, which are difficult to mimic in experimental animal models. Thus, clinical allergy may develop even to extremely weak sensitizing drugs that can be safely used orally, and escape most currently available predictive contact allergy animal models. Clinical studies remain unavoidable for studying factors that may reduce sensitization rates to more acceptable levels. [ABSTRACT FROM AUTHOR]

Different lymphocyte subpopulations were prepared from lymph nodes and spleen obtained from 2.4-dinitrofluorobenzene/Freund's complete adjuvant (DNFB/FCA) sensitized guinea-pigs. The capacity to react in vitro to secondary antigenic (dinitrophenyl- modified syngeneic macrophages, DNP-Mø) stimulation by an increased DNA synthesis, appeared to be restricted to those T cells which lack receptors for lgG-Fc (Tγ- ). We confirmed the existence in this species of an IgG-Fc receptor-positive T-cell subset, which is particularly represented in the spleen (up to 56%). These Tγ+ cells apparently are not of monocyte lineage as they show strong PHA-responsiveness and a lack of non-specific esterase staining, whereas the majority of these cells form RRBC-rosettes. Neither B nor Tγ+ cells appeared to act as suppressor cells in the secondary DNP-Mø-driven proliferative response. [ABSTRACT FROM AUTHOR]

Until now in vitro stimulation tests have failed to detect DNCB contact sensitivity in guinea-pigs sensitized epicutaneously without the use of adjuvants. In this study DNP-conjugates optimally inducing contact sensitivity in vivo were tested for their capacity to detect DNCB contact sensitivity in vitro in a lymphocyte transformation assay. In vivo contact sensitivity measurements in guinea-pigs which had been immunized with different hapten-cell conjugates, showed that (1) living cells should be used for conjugation with the allergen, (2) macrophages are optimal carriers and (3) syngeneity is not required. Therefore, DNFB-coated peritoneal macrophages (viable when conjugated) were used as an antigen for in vitro stimulation. Using this conjugate, a highly reproducible antigen-specific increase in DNA synthesis could be obtained in lymph node lymphocytes from guinea-pigs that had been sensitized to DNCB by epicutaneous application of the allergen without the use of adjuvants. [ABSTRACT FROM AUTHOR]

Rabbit erythrocyte (RRBC) and EAC rosette formation, for detection of T and B lymphocytes respectively, was studied in thymus, lymph nodes and peripheral blood of guinea-pigs. Using two different methods for preparation of RRBC rosette-forming cells (RFC) it was found that, without an incubation period at 37°, a smaller part of the pool of T cells is detected which is particularly sensitive to cyclophosphamide (cy). RRBC rosette formation was studied for 2 weeks after treatment with one high dose of cy and appeared to be minimal in the thymus after 1 week and maximal in the lymph nodes at about day 3. EAC rosette formation was completely blocked by cy treatment. Four days after contact sensitization with 2,4-dinitrochlorobenzene (DNCB) no percentual increase was found in RRBC-RFC, suggesting a parallel increase of B and T cells in stimulated lymph nodes. Cytological screening of the rosettes at day 4 after DNCB showed that of the RRBC-RFC approximately 17 per cent were blast cells, while no difference was detected in the number of blast cells participating in EAC rosette formation as compared to the unstimulated lymph nodes. In contrast to RRBC-RFC the percentage of EAC-RFC in draining lymph nodes is increased after contact sensitization. The mechanism of this increase is discussed. [ABSTRACT FROM AUTHOR]

DNP-guinea-pig erythrocyte rosette-forming cells (RFC), as found after contact sensitization of guinea-pigs with DNCB, were shown to be highly sensitive to pretreatment with cyclophosphamide or X-irradiation, which suggests that these cells are B cells. This finding was confirmed by rosette-blocking experiments using an anti-immunoglobulin serum. It has already been shown that they are not directly related to antibody production. Possibly they represent precursors of antibody -producing cells. An increase in rosette formation, using non-antigenically related erythrocytes, was shown to parallel development of cell-mediated immunity (CMI) in the lymph nodes. These non-specifically induced RFC were also found to be B cells. Data were obtained which make it improbable that either proliferation or influx of B cells during development of CMI plays a major role in this increase. An alternative mechanism is suggested, while a possible role for these non-specifically induced RFC is proposed.

A prospective study of the development of hand eczema was initiated in 86 junior hairdressers and 217 junior nurses. Data obtained at the start of their apprenticeships are presented. None of the junior apprentices presented with hand eczema. but a history of hand eczema was reported by 22 303 (7%) of the students, Almost half of this group (10,22, 45%) could be classified as atopics. Students without a history of hand eczema showed a similar frequency of atopy (17%) as observed in the general population. History of hand eczema was not related In nickel hypersensitivity, as assessed by patch testing. The incidence of nickel hypersensitivity was high in junior hairdressers (26%), compared to junior nurses (12%). Information as to previous contacts with nickel suggested that development of nickel allergy had been promoted by ear-piercing. A lower incidence of nickel hypersensitivity was observed if this potentially sensitizing event had been preceeded by orthodontic treatment with nickel-containing materials. This phenomenon is discussed in terms of orally-induced tolerance. [ABSTRACT FROM AUTHOR]

Irritant reactions to sodium lauryl sulfate were induced on the backs of 20 volunteers by means of patch test occlusion for 24 h. Different concentrations ranging from 0.25% to 2% were used, the lowest concentration being borderline irritant. The skin tests were read at 24, 48 and 72 h. Both the 96 of responding individuals and the intensity of the skin reactions were maximal at 48 h for all test concentrations. It is concluded that irritants may provoke inflammatory reactions which are not completely developed after 24 h. and are thus very similar to allergic patch test reactions. [ABSTRACT FROM AUTHOR]

Irrelevant reactions occurred in 68 of 157 patients (43%) with strong positive patch teat reactions and concomitant weak positive reactions. A lower figure, 13 of 45 (29%) was found in patients with only weak positive patch test reactions (0. 1 > P > 0.05). An allergen eliciting' a weak positive reaction should therefore be retested when a conclusion has to be drawn about the epidermal sensitivity of a patient. [ABSTRACT FROM AUTHOR]

Delayed hypersensitivity reaction in mice was enhanced with various anti-cancer drugs administered at the site of antigenic stimulation during 4 days following sensitization. The immunopotentiating effect of the presented local chemotherapy protocol is thought to result from impairment of a regulatory circuit, with activated suppressor T cells (Ts) as target rather than Ts precursors or Ts-inducing antigen-presenting cells. [ABSTRACT FROM AUTHOR]

Studies were carried out in order to explain the often small contribution of eosinophils to immunologically mediated chronic inflammatory reactions. A chronic inflammation model was used in which large numbers of cosinophils accumulated in the peritoneal cavity of guinea-pigs following repeated intraperitoneal injections with PPD or ovalbumin (OA). Eosinophil accumulation could be strongly suppressed by pre-immunization with a mycobacteria- containing adjuvant, which induces delayed hypersensitivity to the stimulating antigen. Suppression of OA-induced eosinophil accumulation could be obtained in a non-specific way by concomitant delayed hypersensitivity reactions to PPD. The absence of eosinophil accumulation was not related to a lack of relevant antibodies. Histological studies suggested that similar numbers of basophils were locally available in both non-pre-immunized and pre- immunized groups to allow for the attraction of eosinophils by the expression of basophil anaphylactic reactivity. Skin tests and macrophage migration inhibition measurements provided further support for the hypothesis that local lymphokine release may suppress the accumulation of eosinophils in chronic inflammatory lesions. [ABSTRACT FROM AUTHOR]

A chronic inflammation model is described which allows the study of the relationship between the level of specific cell-mediated and humoral immunity to the triggering antigen, and the presence of cells or mediators in the inflammatory exudate. In the present study special attention is paid to the participation of lymphocytes and eosinophils. Normal or FCA-pre- immunized guinea-pigs received repeated intraperitoneal injections with PPD for periods up to 21 weeks. In non-pre-immunized animals the inflammation was characterized by a strong accumulation of eosinophils, whereas only a few lymphocytes were present. In contrast, the FCA-pre-immunized guinea-pigs showed a strong lymphocytic accumulation in the absence of eosinophils. Both peritoneal inflammations were shown to remain dependent on the continuous PPD administration. Therefore, the participation of either lymphocytes or eosinophils could be directly correlated to differences in the specific immune status. Both experimental groups developed similar strong anti- PPD antibody responses as assessed by haemagglutination and passive cutaneous anaphylaxis. Only the FCA-pre-immunized guinea-pigs, however, showed strong cell-mediated immunity. The development of CMI to the continuously administered antigen appears to be a prerequisite for the accumulation of lymphocytes, while it seems to prevent antibody- mediated eosinophil accumulation. [ABSTRACT FROM AUTHOR]

The interference of two simultaneous skin test reactions of intermediate strength has been studied in the guinea-pig, using four different antigens, i.e. ovalbumin, horse cytochrome c, PPD and oxazolone. Skin test reactions were evaluated at 4, 24 and 48 h by measuring three parameters: increase in skin thickness, diameter of erythema and intensity of erythema. When an Arthus reaction was elicited simultaneously with a delayed hypersensitivity (DH) reaction, no effect on the DH reaction was observed. When two simultaneous DH reactions were elicited with different antigens, the risk of interference appeared to be rather small. When, however, the same antigen was used for both skirt tests, suppression of at least one parameter of a DH-reaction was found in almost all experiments. Suppression of one skin test by another one could not be reduced by introducing a larger distance between the two skin tests. As complete inhibition of either of the parameters never occurred, multiple skin testing may allow one to obtain a qualitative impression of the state of delayed hypersensitivity; when, however, reliable quantitative data are needed, the performance of mare than one skin test at a time should be avoided. [ABSTRACT FROM AUTHOR]

Suppressor cells were demonstrated in the spleen of guinea-pigs made specifically unresponsive to dinitrofluorobenzene (DNFB) with dinitrobenzene sulphonic acid (DNBSO3). Transfusion of these cells at the same time as sensitization with DNFB, produced a significant reduction in the immunoblasts proliferating in the draining lymph node 4 days later. Transfusion on the day of skin testing produced no greater suppression of skin reactivity than cells taken from animals made hyporeactive to DNFB by contact with dinitrothiocyanate benzene (DNTB). It is concluded that there are at least two sites that suppressor cells can act. In the case of total unresponsiveness induced by DNBSO3, action is both central and in the periphery. In the case of hyporeactivity induced by DNTB, in which there is no defect in proliferation of T cells in response to antigen, the action of these cells is confined to the periphery. Results of spleen weight studies suggest that suppressor cells homing in the spleen respond by proliferation to epicutaneously applied DNFB. [ABSTRACT FROM AUTHOR]

This report presents the characterization of three mouse monoclonal antibodies (mAb) reactive with the guinea-pig leucocyte common antigen (LCA); CD45 in the human nomenclature. One, IH-l, reacted with LCA on all leucocytes. The other two were more restricted: IH-2 recognized only the 220,000, 210,000 and 195,000 MW isoforms, and IH-4 the 220,000, 210,000 MW isoforms. Both IH-2 and IH-4 reacted with all B cells and all Kurloff cells [the putative guinea-pig natural killer (NK) cell]. IH-2, but not IH-4, reacted with monocytes and macrophages. Neither reacted with neutrophils. Most thymocytes expressed low levels of the IH-2 and IH-4 epitopes, with those expressing high levels located predominantly within the medulla. Most (90%) CD4+ T cells from newborn guinea-pigs expressed high levels of the IH-2 and IH-4 epitopes; this percentage decreased with age to 70% in 2-year-old animals. We have demonstrated that CD4+ T cells which express low levels of the IH-2 epitope also express low levels of the IH-4 epitope. CD8+ T cells can be divided into two subsets by IH-4 but not IH-2. The reactivities of IH-2 and IH-4 are remarkably similar to those of human anti- CD45RB and anti-CD45RA antibodies respectively. Analogies with man and other species suggest important functional differences for subpopulations of guinea-pig T lymphocytes defined by anti- CD45R antibodies. [ABSTRACT FROM AUTHOR]

A single systemic dose of cyclophosphamide (CY) has been shown to enhance cellular immunity in a variety of antigen models. The immunoenhancing effects of CY have been attributed to its ability to selectively abrogate suppressor cell function. Previous studies from our group have demonstrated that local administration of distinct cytostatic drugs at the sensitization site can induce a similar enhancement of delayed-type hypersensitivity as systemic CY, with the obvious advantage of avoiding systemic side-effects. In the present study we investigated the effects of local administration of an optimally immunopotentiating dose of the active CY-derivative Z 7557 and, in selected experiments, of etoposide (VP- 16) and systemic CY on mononuclear cells in draining lymph nodes. Whereas CY caused a long-lasting and marked depletion of B-cell areas, locally administered Z 7557 and VP- 16 relatively spared B cells and even induced an increase in B- and 1-cell numbers in (keyhole limpet haemocyanin-) sensitized mice. At Day 4 the CD4JCD8 ratio was slightly reduced in drug- treated mice. Interestingly, drug treatment reduced the proportion of interdigitating cells staining with the monoclonal antibodies NLDC-145 and MIDC-8. Upon isolation, dendritic cells (DC) from sensitized, Z 7557-treated mice showed longer dendritic protrusions and an enhanced accessory cell function compared to DC from saline-treated controls. These findings suggest that immunoenhancing effects of cytostatic drugs may occur via an effect on DC. [ABSTRACT FROM AUTHOR]

A monoclonal antibody is described which reacts with monocytes and macrophages of the guineaspig. The antibody recognizes a cytoplasmic component in the majority of tissue macrophages, as detected by histochemical analysis. It reacts positively with all macrophages in lymphoid organs, including Tingible body macrophages. In addition it recognizes the cells of Von Kupifer in the liver, and macrophages in bone marrow and peritoneal cavity. In comparison to other monoclonal antibodies that have been described in the guinea-pig, MR-1 shows a unique tissue distribution. The antibody is a useful tool to discriminate macrophages by immunohistochemical methods. [ABSTRACT FROM AUTHOR]

Guinea-pig lymphokines were shown to stimulate the migration of eosinophils from capillary tubes. Eosinophil migration stimulatory activity was produced by Freund's complete adjuvant (FCA)- sensitized lymph node or peritoneal exudate lymphocytes in the presence of purified protein derivative (PPD), as well as by Con A-stimulated lymph node lymphocytes. Like murine and human eosinophil-stimulation promoter lymphokine (ESP), the guinea- pig lymphokine activity is T cell-derived, non-dialysable and resistant to heating at 56°. In contrast to the migration inhibition factor (MIF) which could be adsorbed by macrophages, eosinophil migration stimulatory activity could not be removed by pre- adsorption to macrophages or eosinophils. [ABSTRACT FROM AUTHOR]

Guinea-pigs have been sensitized with DNCB, DNP-guinea-pig serum and DNP-guinea-pig erythrocytes. Four, six and ten days after sensitization numbers of rosette-forming cells with DNP-erythrocytes were estimated in the draining lymph nodes. It was found that the numbers of DNP-erythrocyte RFC did not correlate with humoral immunity as estimated by haemagglutination assay, but did so with cell- mediated immunity estimated as delayed-type hypersensitivity. On the other hand histological examination of the draining lymph nodes gave no clear confirmation of such a relationship. Experiments on the antigenic specificity of RFC showed that after DNCB sensitization a clear increase iii oxazolone-erythrocyte RFC and SRBC RFC can be found. These latter data suggest formation of RFC to be at least partly a non- specific phenomenon. [ABSTRACT FROM AUTHOR]

Low molecular weight factors, derived from head and neck carcinomas (H/N ca LMWFs), exert a serious inhibitory effect on monocyte chemotaxis both in vivo and in vitro. This effect can be neutralized by the treatment of the factors with three different monoclonal antibodies to P15E, one of the structural envelope proteins of murine leukemia virus (MuLV). In this study, we report the effect of the H/N ca LMWFs on the delayed type hypersensitivity responsiveness in mice: the factors significantly inhibited the 24 hour DNFB skin reaction. These effects of the H/N ca LMWFs were again neutralizable by antibodies to P15E. Additional experiments showed that the H/N ca LMWFs had equal suppressive effects on toxic skin reactions to croton oil, and this indicates that the P15E-like H/N ca LMWFs exert their effect by aspecific mechanisms. [ABSTRACT FROM AUTHOR]

Breast cancer resistance protein (BCRP) is a recently described member of the ATP binding cassette transporter superfamily. It has been shown to confer resistance to mitoxantrone, topotecan, doxorubicin and daunorubicin in human tumour cell lines. We describe a study of BCRP expression in blast cells derived from 20 patients with acute myeloid leukaemia (AML). Twelve samples were from patients who had received previous cytotoxic therapy. BCRP expression was measured by immunocytochemistry using the BXP-34 monoclonal antibody. In vitro drug sensitivity was assessed using the methyl thiazol tetrazoliumbromide assay. BCRP expression varied between patients, and six out of 22 (27%) samples had > 10% cells staining positively (median 37%, range 13-95%). BCRP positivity was seen in both de novo samples and those from previously treated patients. There was a marked variation in the effect of all drugs tested between patients. Although there was no correlation between BCRP positivity and the effect of mitoxantrone, topotecan or doxorubicin, the median daunorubicin LC(50) value of BCRP(+) cells was fourfold higher than that of BCRP- cells (0.89 micromol/l compared with 0.21 micromol/l, P < 0.05). These results suggest that BCRP may be involved in resistance to the agents commonly used in AML and may explain some of the anomalous results found when studying other membrane transporters, such as P-gp or MRP.

Fanconi anaemia (FA) is an autosomal recessive disease strongly predisposing to bone marrow failure and acute myeloid leukaemia (AML). Four FA genes, corresponding to complementation groups A, C, F and G, have been cloned, but the molecular functions of the corresponding proteins are unknown. The high risk of AML in FA patients suggests that the 'FA pathway' helps to prevent AML in non-FA individuals. We examined 10 AML cell lines, as well as primary cells from 15 AML patients representing the French-American-British subclasses M1-M5a, for possible deficiencies in the 'FA pathway'. Cellular lysates were analysed for the presence of the FA proteins FANCA, FANCC, FANCF and FANCG, as well as the complexes reported to be formed between these proteins, using immunoprecipitation and Western blot analysis. Aberrant protein profiles were observed in five of the 10 cell lines and in 11 of the 15 primary AML samples. Aberrations, that included absence or reduced presence of FA proteins and/or their complexes, were noted in the subclasses M1-M4, but not in M5a (n = 3). Our results suggest that a significant proportion of general AML is characterized by a disturbance of the 'FA pathway' that may represent an early event in the development of this type of leukaemia.

The in vitro intracellular daunorubicin accumulation (IDA) of blast cells from 69 patients with newly diagnosed acute myeloid leukaemia (AML) was correlated with the expression and functional activity of the multidrug resistance (MDR) proteins, P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP). An inverse and significant association was found between IDA and Pgp-related efflux activity (r = -0.31, P = 0.01) and also MRP (r = -0.25, P = 0.04) but not with LRP (r = -0.13, P = 0.28). Coexpression of the MDR proteins had an additive effect in further lowering of IDA levels, suggesting that the clinical MDR phenotype is dependent on the sum of multiple MDR factors available to the leukaemic cell. Thus, the median IDA of leukaemic cells without any MDR proteins was significantly higher than that of blasts carrying two MDR proteins (0.466 vs. 0.296, P = 0.046). Seven patients with no expression of Pgp, MRP and LRP still had low IDA levels, suggesting the presence of efflux MDR mechanisms other than those studied. The relation of IDA to clinical parameters known to be associated with poor prognosis, such as age, secondary AML, karyotype, peripheral blood blast and CD34 counts, was also studied, but no significance was found on multifactorial analysis. There was a non-significant trend for earlier relapse in patients with low IDA levels (leukaemia-free survival of 16.3 months compared with 21.1 months in patients with high IDA levels). Our data suggest that, while the IDA assay is a quick and relatively easy test for the combined efflux MDR phenotype, it is unable to detect other MDR mechanisms, such as LRP, which may be important to the clinical outcome of patients with AML.

P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance associated protein (MRP) expression and the blast cells' intracellular daunorubicin accumulation (IDA) were evaluated in 96 previously untreated cases of de novo acute non-lymphocytic leukaemia (ANLL). 47/96 patients (49%) were classified as PGP+ 44/ 96 (46%) as LRP+, and 8/96 (8%) as MRP+. The more frequent MDR clusters were PGP-/LRP-/MRP- (32/96 cases, 33%) and the PGP+/LRP+/MRP- (27/96 cases, 28%) followed by PGP+/LRP-/MRP- (15/96 cases, 16%) and PGP-/LRP+/MRP- (14/96 cases, 14%). A favourable karyotype was observed more frequently in PGP- and LRP-cases. A highly significant correlation was found between either PGP or LRP overexpression and leukaemic blast cell IDA. All the patients received standard induction and consolidation treatments containing MDR-related (idarubicin, mitoxantrone, etoposide) and other (arabinosyl cytosine) drugs. Multivariate analysis showed that PGP overexpression was significantly associated with a poor response to treatment, both in terms of primary resistance or shorter survival. Other independent prognostic factors were age and cytogenetics. LRP overexpression did not reach statistical significance, although for LRP+ cases the trend was unfavourable. Due to small numbers, no conclusion could be made regarding MRP overexpression, but 5/8 cases showed unfavourable karyotypic abnormalities, 8/8 had a defective IDA and 6/8 failed to achieve remission. This study showed that both PGP and LRP overexpression are common features in de novo ANLL at onset whereas MRP overexpression is more rare. It suggested that overexpression of one of the MDR related proteins was associated with a defective IDA, and confirmed that, in addition to age and cytogenetics, PGP retains an independent prognostic value. It also suggested that LRP did not affect clinical outcome when patients were treated with idarubicin or mitoxantrone and arabinosyl cytosine.

We investigated the role of the drug resistance-related proteins LRP, MRP and Pgp and the apoptotic suppressor, bcl-2, in relation to other clinical characteristics, with respect to response and survival in 91 patients with newly diagnosed AML, treated with standard chemotherapy. Multivariate analysis showed that poor response to chemotherapy was associated with increasing age (P=0.0004), LRP expression (P=0.0001) and Pgp function (P=0.015). The significant predictors of both leukaemia-free survival (LFS) and overall survival (OS) were LRP (LFS, P=0.01; OS, P=0.0001), Pgp function (LFS, P=0.0001; OS, P=0.0003) and cytogenetic abnormalities (LFS, P=0.0001; OS. P=0.0005). Patients with the lowest expression of LRP and Pgp function and favourable karyotype (group I) had an LFS of 30.2 months compared to 8 5 months in the group with the highest expression of LRP and Pgp and poor prognosis karyotype (group III, P=0.002). OS decreased from 75.4 months in group I to 7.9 months in group III patients (P <0.0001). Neither MRP nor bcl-2 were significantly associated with chemotherapy response and survival. Correlations were found between increasing expression of LRP and older age (P=0.05) and an unfavourable karyotype (P=0.005), but these variables were independent of each other in analysis of treatment response and patient survival. Our findings suggest that both LRP and Pgp are clinically relevant drug-resistance proteins and it may be necessary to modulate both LRP and Pgp functions in order to reverse the multidrug resistance phenotype in AML.

The purpose of the present study was to evaluate whether intermittent exposure to a constant dose of doxorubicin selects for multidrug resistance (MDR) in RPMI 8226 human myeloma cells and, if so, to determine the molecular mechanism. In an attempt to approximate clinical doxorubicin treatment in vitro, cells were exposed to a fixed dose of doxorubicin for 4 d alternating with growth in drug-free medium for 17 d. An MDR subline emerged, termed 8226/DOXint5, which was 3-4-fold resistant to doxorubicin, etoposide and m-AMSA, and 1.6-fold resistant to vincristine. Sensitivity to docetaxel, melphalan and cisplatin was normal. Verapamil normalized vincristine sensitivity but had little effect on resistance to the other agents. Cellular uptake and retention of daunorubicin and vincristine were reduced by approximately 10%. The 8226/DOXint5 cells showed diminished DNA topoisomerase IIalpha expression and increased expression of the multidrug resistance protein MRP. Expression of MDR1/P-glycoprotein was not detected. Immunostaining showed 70% of the cells to over-express the lung-resistance protein LRP. This new MDR myeloma cell line may prove to be a useful model for the development of strategies to overcome low-level, multifactorial MDR, which might be a common phenomenon in clinical myeloma treated with doxorubicin.