Effect of partially modified retro-inverso analogues derived from C-reactive protein on the induction of nitric oxide synthesis in peritoneal macrophages.

1. The ability of three modified tetrapeptides, representing fragments of the C-reactive protein (CRP) sequence and stabilized in the first peptide bond by retro-inverso modification, to affect the secretion of nitric oxide (NO) was studied in macrophages of BALB/c mice. 2. These tetrapeptides, resembling the aminoacid sequence of tuftsin (CRP 1, H-gThr-(R,S)mLys-Pro-Leu-OH, ITF 1192; CRP II, H-gGly-(R, S)mLys-Pro-Arg-OH, ITF 1127; CRP III, H-gThr-(R,S)mLys-Pro-Gln-OH. ITF 1193), were able to induce NO synthesis by peritoneal macrophages in a dose-dependent manner; the most stimulating dose was 1000 ng ml-1 for CRP II and 100 ng ml-1 for CRP I and CRP III. NO synthesis was not strictly dependent on lipopolysaccharide (LPS) activation. 3. The enhanced effect of retro-inverso CRP-related analogues on the expression of iNOS (inducible NO synthase) was confirmed by higher levels of iNOS activity in the cytosol and by the increase in iNOS protein, as evaluated by Western blot analysis, in macrophages stimulated by CPR compared with untreated ones. 4. The production of NO by retro-inverso CRP-peptide analogues was significantly inhibited by dexamethasone (20 microM), NG-monomethyl-L-arginine (L-NMMA) (500 microM) and pyrrolidine dithiocarbamate (PDTC) (100 microM). 5. Retro-inverso CRP-peptide analogues stimulated macrophages to produce high levels of interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) in the presence of LPS. 6. Retro-inverso CRP-peptide analogues stimulated NO synthesis by the enhancement of endogenously produced IL-1 and TNF-alpha, as the treatment of peritoneal macrophages with LPS in the presence of neutralizing anti-IL-1 and anti-TNF monoclonal antibodies (mAbs) reduced retro-inverso analogue-induced NO secretion. Data indicate a predominant role for IL-1 alpha in the induction of NO secretion by retro-inverso analogues. 7. These results suggest that retro-inverso CRP derived analogues act as costimulators of NO and cytokine synthesis in macrophages. The mechanisms by which they cause iNOS induction appear to be strongly dependent on the activation of nuclear factor-kappa B (NF-kappa B).

Rational design of a new C-myristylamido peptide exerting potent and selective PKC inhibitory activity.

A 3D model of the catalytic domain of PKC was built based on the X-ray structure of the homologous PKA enzyme. The two enzymes were found to have similar general architecture although differing for the number of negatively charged clusters and their location near the phosphorylation site. These differences were consistent with the charge requirements deduced from the consensus sequence of PKC and PKA substrates. A Myristyl Binding Site (MBS) was found in the PKC model between helix C and sheets 8 and 9. The identification of this MBS allowed the rationalization of the results obtained with N-myristoylated peptide inhibitors and, above all, the design of ITF1671 (H-RFARKGALRQKN-CONH-Myr), a new C-myristylamido peptide, which exerted one of the most potent inhibitory activity against PKC and PKM known to-date.

Different cytotoxic activity and intracellular fate of an anti-CD5-momordin immunotoxin in normal compared to tumour cells.

We investigated the different sensitivity of peripheral blood mononuclear cells (PBMC) and human T cell leukaemias (Jurkat and CEM) to an anti-CD5-momordin immunotoxin. In a short-term assay, the immunotoxin displayed different cytotoxic activity on normal and tumour cells: for leukaemic cell lines an incubation time of 72 h was necessary for the immunotoxin to reach the IC50 of 41-53 pM, compared to the 1 h sufficient for 6 pM immunotoxin to inhibit 50% of PBMC protein synthesis. In a long-term clonogenic assay (15 days), the immunotoxin demonstrated a comparable efficacy of clonogenic cell killing for both cell types. We investigated the immunotoxin internalization pathway by a flow-cytometric method and our data seem to indicate that the molecules meet a different intracellular fate in the two cell populations. It may be assumed that the low cytotoxic activity of immunotoxins on tumour cells, detected in the short-term assay, is due to inefficient delivery to their cytoplasmatic target, while a longer exposure of the cells to the immunotoxin promotes adequate intracellular distribution.

Expression in Escherichia coli, purification and functional activity of recombinant human chaperonin 10.

We have recently reported the cloning of a cDNA coding for a stress inducible human chaperonin 10. The protein was shown to possess 100% identity with the bovine homologue and a single amino acid replacement (glycine to serine at position 52) compared to rat chaperonin 10. Here we report the heterologous expression of human chaperonin 10 in Escherichia coli, its purification and its functional characterization. The recombinant protein was purified to homogeneity as judged by different analytical techniques, and mass spectrometry analysis showed a MW of 10,801 Da in agreement with the predicted sequence. This molecular weight accounts for a protein which is not modified post-translationally. In fact, natural rat chaperonin 10 has been shown to be acetylated at the N-terminus, a feature suggested to be important for targeting and functional activity. Here we show that recombinant human chaperonin 10 is fully active in assisting the chaperonin 60 GroEL in the refolding of denatured yeast enolase, thereby showing that, at least in the present system, post-translational acetylation is not necessary for its activity.

Cochaperonins are histone-binding proteins.

Cochaperonins (cpn10) assist chaperonins (cpn60) in mediating folding of polypeptide substrates in an ATP-dependent reaction. Moreover, they have been shown to be secretory products of living cells and to perform discrete biological activities without the need to interact with cpn60. Here, we have investigated the possible existence of cellular cpn10 binding sites that could mediate such activities. For this purpose, we performed binding studies with iodinated cpn10 on whole cells and on electrophoretically separated eukaryotic cell lysates. The former studies yielded negative results, whereas in the latter binding to several proteins was detected. These proteins were identified as being histones. Binding was observed to all core histones (H2A, H2B, H3 and H4) and, although weaker, to the linker histone H1 as well. These results show that cpn10 are histone-binding proteins.

Identification and cloning of human chaperonin 10 homologue.

We have identified a heat-shock-inducible 10 kDa protein in the human hepatoma cell line HepG2. The total RNA extracted from the heat-shocked cells was amplified by reverse transcription PCR (polymerase chain reaction) using 21 5' and 18 3' oligonucleotides of rat cpn10 (chaperonin10) cDNA as primers. Sequencing of the above PCR fragment showed a very high homology between human, bovine and rat cpn10 cDNA. The predicted amino acid sequence revealed a 100% identity with the bovine homologue.

The macrophage-activating tetrapeptide tuftsin induces nitric oxide synthesis and stimulates murine macrophages to kill Leishmania parasites in vitro.

The macrophage-activating tetrapeptide tuftsin was able to activate, in a dose-dependent manner, murine macrophages to express nitric oxide (NO) synthase and to produce NO. Tuftsin required lipopolysaccharides for the optimal induction of NO production and synergized with gamma interferon in the induction of NO synthesis. Tuftsin-dependent NO production was sensitive to inhibition by dexamethasone and the NO synthase specific inhibitor LGN-monomethylarginine (L-NMMA). Murine peritoneal macrophages activated by tuftsin were able to kill the amastigotes of the intracellular protozoan parasite Leishmania major in vitro.

Delivery of saporin to human B-cell lymphoma using bispecific antibody: targeting via CD22 but not CD19, CD37, or immunoglobulin results in efficient killing.

A panel of bispecific F(ab')2 antibodies (BsAb) have been constructed for delivering the ribosome-inactivating protein saporin to human B cell lymphoma. Each derivative was prepared with specificity for saporin and CD19, CD22, CD37, or immunoglobulin. In vitro studies measuring inhibition of [3H]leucine uptake by cultured Daudi and Raji cells demonstrated that, despite all BsAb capturing saporin on the cell surface, BsAb targeting through CD22 were far more cytotoxic than those functioning via CD19, CD37, or surface immunoglobulin. This exceptional activity of the CD22-specific BsAb appears to derive from its ability to deliver and accumulate saporin inside the target cells. Further studies showed that four CD22-specific BsAb all performed with equal potency and were able to increase saporin toxicity (50% inhibitory concentration) up to 1000-fold, from 2 x 10(-7) M to 2 x 10(-10) M. Pairs of anti-CD22 BsAb which recognized different nonblocking epitopes on the saporin molecule were able to bind saporin more avidly to the target cell and, as a consequence, increased cytotoxicity by at least an additional 10-fold, resulting in 50% inhibitory concentration for protein synthesis of 2 x 10(-11) M. These results suggest that selected combinations of BsAb which bind cooperatively to a toxin and the cell surface may provide an efficient way of delivering toxins to unwanted cells in patients.

In vitro and in vivo properties of an anti-CD5-momordin immunotoxin on normal and neoplastic T lymphocytes.

An anti-CD5 monoclonal antibody (mAb) was linked to the plant toxin momordin, a type-1 ribosome-inactivating protein purified from Momordica charantia. The in vitro cytotoxicity of the immunotoxin was evaluated as the inhibition of protein and/or DNA synthesis on isolated peripheral blood mononuclear cells (PBMC) and on human T cell leukemia Jurkat. The potency of the immunotoxin on PBMC was very high (IC50 = 1 - 10 pM) and was not affected by blood components. The conjugate was also very efficient in the inhibition of the proliferative response in a mixed lymphocyte reaction (IC50 = 10 pM). Moreover, the in vitro performance of the immunotoxin compared favorably with those reported for other anti-CD5-based immunoconjugates containing ricin A chain. The in vivo activity of the immunotoxin was assessed in the model of nu/nu mice bearing Jurkat leukemia. A significant inhibition of the tumour development (80%, P < 0.01) in the animals treated with immunotoxin was observed. Taken together, the in vitro and in vivo results suggest that the anti-CD5-momordin conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5-positive leukemias and lymphomas.

Expression and activity of pre-dianthin 30 and dianthin 30.

Dianthin 30 is a ribosome inactivating protein (RIP 1) found in different tissues of the carnation (Dianthus caryophyllus). Recently we have isolated and sequenced a cDNA clone from a lambda gt11 expression library [Legname et al. (1991) Biochim. Biophys. Acta 1090, 119-122]. Here we describe specific PCR amplifications of either the full length pre-dianthin 30 or dianthin 30, the mature polypeptide lacking the 23 amino acid signal peptide. In vitro expression of both proteins in reticulocyte lysate generated products of the expected molecular weight. Moreover, the activity of both proteins has been evaluated confirming the characteristics of the natural product. A first attempt to produce recombinant dianthin 30 in Escherichia coli is described.

A comparison of anti-lymphocyte immunotoxins containing different ribosome-inactivating proteins and antibodies.

Immunotoxins were prepared with several single-chain ribosome-inactivating proteins (RIPs type 1) and with the A-chain of ricin linked to the F(ab')2 fragment of sheep anti-mouse IgG. The cytotoxic activity of these conjugates was tested on human lymphocytes pretreated with an anti-CD3 murine MoAb. The immunotoxins inhibited DNA synthesis in phytohaemagglutinin (PHA)-stimulated lymphocytes with IC50S (concentrations causing 50% inhibition) ranging from 8.9 x 10(-13) to 5.7 x 10(-11) M (immunotoxins containing dianthin 32, saporin, pokeweed antiviral protein from seeds (PAP-S), bryodin, momordin, momorcochin, and trichokirin), 1 x 10(-8) M (immunotoxin containing gelonin) and 5 x 10(-9) M (immunotoxin containing ricin A-chain). The immunotoxin containing saporin linked to the anti-mouse IgG F(ab')2 fragment was also highly toxic to human lymphocytes pretreated with anti-CD2, -CD3, -CD5 and -CD45 MoAbs, with IC50S less than or equal to 10(-11) M. Immunotoxins were prepared also with saporin linked to MoAbs against various CD antigens. The immunotoxin prepared with the anti-CD3 antibody had the highest specific cytotoxicity to human lymphocytes.

Modulation of IL-2, IFN-gamma, TNF-alpha and IL-4 production in mice of different ages by thymopentin.

The effect of an immunomodulator drug thymopentin (TP5) on the production of various cytokines (IFN-gamma, IL-2, IL-4, TNF-alpha) in mice of different ages has been studied. TP5 enhanced IL-2, TNF-alpha and IFN-gamma production but reduced the IL-4 secretion by splenocytes from aged mice (greater than 120 week old) in vitro. However, it had no effect on the IL-2, IFN-gamma, TNF-alpha or IL-4 production by splenocytes from young and adult mice. TP5 injected subcutaneously was able to induce high levels of IL-2 production by splenocytes from all groups of mice. The TP5 effect on TNF-alpha and IFN-gamma was similar, even though it was significant only in old mice. Furthermore, TP5 was able to significantly reduce IL-4 production in old mice, which normally produced high levels of this cytokine after mitogen stimulation. Since it has been observed in the mouse that the Th1 cells secrete IFN-gamma and IL-2, whereas the Th2 cells preferentially produce IL-3, IL-4 and IL-5, these results indicate that the immunopotentiatory activity of TP5 is due to the preferential up-regulation of Th1 cells.

Initial experience in treating human lymphoma with a combination of bispecific antibody and saporin.

Results are presented showing the use of bispecific F(ab')2 antibodies (bsAbs) in the delivery of saporin for the treatment of 2 human B-cell malignancies. BsAbs delivering saporin through CD22, but not through CD19, were effective at inhibiting the uptake of [3H]leucine by Daudi and Raji cells. Furthermore, a combination of 2 anti-CD22 bsAbs, selected to bind simultaneously to saporin, bound saporin 20 times more avidly and inhibited protein synthesis far more efficiently than any single bsAb. In the first patient, with end-stage chronic lymphocytic leukaemia (CLL), treatment with 10 mg of saporin complexed to 100 mg of anti-CD19 bsAb over 43 days showed no therapeutic effect. In contrast, the second patient, with end-stage non-Hodgkin's lymphoma (NHL), given 5 mg of saporin complexed with a pair (50 mg) of anti-CD22 bsAbs over 15 days showed a marked clinical response, including complete clearance of tumour from the blood, clearance of ascites and shrinkage of tumour masses. Neither patient experienced any toxic side-effects, either during or after treatment. However, the second patient developed a strong anti-mouse Fab (HAMA) response 28 days after the treatment started. No anti-saporin response could be detected.

Anti-CD2 monoclonal antibodies and calcium ionophore A23187 modulate lytic activity in CD4+ and CD8+ alloreactive clones.

Alloreactive cytolytic clones can be modified in terms of their lytic specificity by several agents. We have evaluated the effects of anti-CD2 and anti-CD3 mAb and the calcium ionophore A23187 on CD4+ and CD8+ allospecific cytolytic clones. All these agents can modify the specificity of clones although the range of targets lysed depends on the agent used and subtype of the clone. Inhibition studies suggest that surface structures that are involved in non-specific cell-cell interaction processes play a role in this non-MHC-restricted cytolysis.

Stepwise activation of T cells. Role of the calcium ionophore A23187.

The calcium ionophore A23187, at a concentration of 1 microgram/ml, is able to stimulate proliferation of freshly isolated peripheral blood lymphocytes, CD4+-enriched cells, or CD8+-enriched cells as measured by [3H]thymidine incorporation. This proliferation is accompanied by an increase in interleukin 2 (IL-2) receptor expression but not by a detectable up-regulation in (IL-2) production or the development of cytotoxicity. Proliferation can be blocked by anti-CD3, CD4, or CD8 monoclonal antibodies, but not by anti-Tac. If CD8+-enriched cells are activated for 3 days with A23187 and the blasts present on day 3 are sorted and returned to culture, they rapidly develop cytolytic activity in the presence of recombinant IL-2 but not recombinant interferon-gamma. CD4+ enriched cells, after activation with A23187, do not become cytotoxic in the presence of either recombinant IL-2 or recombinant interferon-gamma. These findings permit study of the stepwise maturation of T cells in this alternative pathway by using "minimal signals" that do not, by themselves and as used in these studies, stimulate precursor Tc to mature to full effector cytotoxic function. These findings are consistent with the model that A23187 drives T cells only part way along a pathway of maturation and that an additional second signal must be given to effect maturation of cytotoxic status.

Long-term growth of lymphokine-activated killer (LAK) cells: role of anti-CD3, beta-IL 1, interferon-gamma and -beta.

Peripheral blood lymphocytes (PBL) cultured in interleukin 2 (IL 2)-containing medium in conventional tissue culture develop the ability to lyse fresh tumor cells; such cells are referred to as lymphokine-activated killer (LAK) cells. LAK activity peaks by day 5 of culture and declines rapidly thereafter. We studied culture conditions and signals that allow for long-term culture and expansion of cells with LAK activity. By culturing cells at relatively low densities and regularly replenishing medium and recombinant IL 2 (r-IL 2), LAK function is significantly higher as compared with short-term cultures, and remains present for at least 21 days while cell numbers undergo an average 100-fold expansion. By activating these cultures with anti-CD3 (OKT3) monoclonal antibody and r-IL 2, an approximately 1000-fold expansion in the cell number is obtained with maintenance of comparable levels of LAK activity. The exogenous addition of beta interleukin 1 (beta-IL 1), interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma) can augment the lytic activity of cell populations expanded by anti-CD3 plus r-IL 2. These approaches may enable the in vitro generation from individual donors of much greater numbers of LAK cells for adoptive immunotherapy than can now be obtained with the 3 to 5 day in vitro culture systems.

Cytotoxic T lymphocytes: blocked differentiation at the 'poised' stage.

The effect of different concentrations of phorbol myristate acetate (PMA) on the development of cytotoxic cells was studied. PMA was selectively able to prevent the development of cytotoxic cells in a mixed leucocyte culture, while allowing the responding cells to proliferate. The higher concentration of PMA (10(-5)M) blocked both direct cytotoxicity and lytic activity in the presence of lectin, while the lower concentration (10(-8) M) only prevented direct lytic function. The removal of PMA and subsequent addition of recombinant interleukin 2 (IL-2) or IL-2-containing supernatants effectively reversed the effect of PMA with recovery of antigen-specific lytic function of cells treated with 10(-8) M, while cells treated with 10(-5)M PMA only recovered lectin-dependent cytotoxic ability.

Role of CD2 and IL 1 beta in T cell responsiveness to IL 2.

We have evaluated the effects of an anti-CD2 monoclonal antibody (moAb), Leu-5b, on inhibition of lymphocyte responsiveness. Given previous reports that anti-CD2 moAb prevent production of interleukin 2 (IL 2), as well as up-regulation of IL 2 receptor, we chose a test system in which lymphocytes were activated by exogenously added recombinant IL 2 (rIL 2). Our studies thus allowed us to evaluate other possible mechanisms by which anti-CD2 moAb could act. We found that Leu-5b not only blocked the proliferative response of human peripheral blood leukocytes (PBL) to IL 2, but also the development of cytotoxicity as assayed in lectin-dependent cytotoxicity (LDCC). Addition of recombinant interleukin 1 beta (rIL 1 beta) reconstituted the proliferative response in the presence of Leu-5b but did not allow development of cytotoxicity. Our findings are consistent with the concept that Leu-5b delivers a signal to IL 2-responsive cells that prevents their subsequent proliferation and development of cytotoxicity, but that only the former event, i.e., proliferation, is reconstituted by the addition of IL 1 beta.

Adenosine induced production of a soluble factor affecting lymphocyte activation.

We show that a brief exposure of human peripheral blood mononuclear cells (PBMC) to adenosine or to theophylline results in a mitomycin C resistant regulatory activity. Adenosine induced suppression is also detectable in a lymphocyte subpopulation (T4+ enriched, originally described as helper inducer) resistant to the theophylline induced loss of capacity to form spontaneous rosettes with sheep erythrocytes (TTR). This activity is apparently dependent on the production of a soluble factor(s) since supernatants from adenosine treated TTR (SnA) exert a significant inhibition on the proliferative response of resting lymphocytes. On the contrary SnA increases the concanavalin A (ConA) preactivated lymphocytes proliferation. Similar results are detectable on the proliferative response in the mixed lymphocyte reaction (MLR). Perhaps these effects are related to different Interleukin 2 (Il 2) receptor expression on the cell surface of the resting and preactivated populations. A slow moving band corresponding to a protein of Mr of 64,500 and isoelectric point 7.6 is present in SnA. Only a slight Il 2 activity is detectable either in SnA and in control supernatant (SnC). These findings suggest that SnA may be a dynamic regulator of the early stages of lymphocyte activation.