Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax.

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.

The 'aromatic patch' of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors.

The role of the functional architecture of the human acetylcholinesterase (HuAChE) active centre in accommodating the non-covalent inhibitors tacrine and huperzine A, or the carbamates pyridostigmine and physostigmine, was analysed using 16 mutants of residues lining the active-centre gorge. Despite the structural diversity of the ligands, certain common properties of the complexes could be observed: (a) replacement of aromatic residues Tyr133, Tyr337 and especially Trp86, resulted in pronounced changes in stability of all the complexes examined; (b) effects due to replacements of the five other aromatic residues along the active-centre gorge, such as the acyl pocket (Phe295, Phe297) or at the peripheral anionic site (Tyr124, Trp286, Tyr341) were relatively small; (c) effects due to substitution of the carboxylic residues in the gorge (Glu202, Glu450) were moderate. These results and molecular modelling indicate that the aromatic side chains of residues Trp86, Tyr133 and Tyr337 form together a continuous 'aromatic patch' lining the wall of the active-centre gorge, allowing for the accommodation of the different ligands via multiple modes of interaction. Studies with HuAChE mutants carrying replacements at positions 86, 133 and 337 indicate that the orientations of huperzine A and tacrine in the HuAChE complexes in solution are significantly different from those observed in X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). These discrepancies may be explained in terms of structural differences between the complexes of HuAChE and TcAChE or, more likely, by the enhanced flexibility of the AChE active-centre gorge in solution as compared with the crystalline state.

Function of the c-Myc antagonist Mad1 during a molecular switch from proliferation to differentiation.

Mad-Max heterodimers have been shown to antagonize Myc transforming activity by a mechanism requiring multiple protein-protein and protein-DNA interactions. However, the mechanism by which Mad functions in differentiation is unknown. Here, we present evidence that Mad functions by an active repression mechanism to antagonize the growth-promoting function(s) of Myc and bring about a transition from cellular proliferation to differentiation. We demonstrate that exogenously expressed c-Myc blocks inducer-mediated differentiation of murine erythroleukemia cells without disrupting the induction of endogenous Mad; rather, high levels of c-Myc prevent a heterocomplex switch from growth-promoting Myc-Max to growth-inhibitory Mad-Max. Cotransfection of a constitutive c-myc with a zinc-inducible mad1 results in clones expressing both genes, whereby a switch from proliferation to differentiation can be modulated. Whereas cells grown in N'N'-hexamethylene bisacetamide in the absence of zinc fail to differentiate, addition of zinc up-regulates Mad expression by severalfold and differentiation proceeds normally. Coimmunoprecipitation analysis reveals that Mad-Max complexes are in excess of Myc-Max in these cotransfectants. Moreover, we show that the Sin-binding, basic region, and leucine zipper motifs are required for Mad to function during a molecular switch from proliferation to differentiation.

Structural modifications of the omega loop in human acetylcholinesterase.

Conformational mobility of the surface omega loop (Cys-69-Cys-96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen-bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the omega loop in the HuAChE is not involved in large lid-like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp-86 supporting the proposed coupling between the structure of the omega loop and the positioning of the Trp-86 indole moiety, in catalytic activity and in allosterism.

Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.

Mechanism of target cell recognition by CD3- LGL. I. Development of a monoclonal antibody to a K562-associated target cell antigen.

In an attempt to identify the target recognition molecule(s) involved in the interaction between CD3- large granular lymphocyte (LGL) and a tumor cell target, monoclonal antibodies (mAb) to NK-susceptible K562 tumor cell membrane glycoproteins were developed. After screening by ELISA for reactivity to K562 membrane glycoproteins, two monoclonal antibodies were identified (mAb 35 and mAb 36). One of the monoclonal antibodies (mAb 36) was found to inhibit conjugation between LGL and K562 target cells and also to inhibit lysis of K562 by LGL. Upon further testing, mAb 36 also inhibited the binding between LGL and other NK-susceptible target cells, e.g., Daudi and Molt 4. In contrast, mAb 35, even though binding to K562, did not inhibit the binding of LGL to tumor targets and therefore was used as an isotype control. When mAb 36 was utilized as an affinity matrix, bound proteins specifically inhibited CD3- LGL-K562 conjugation. Experiments involving tunicamycin treatment of tumor target cells demonstrated that mAb 36 recognized a carbohydrate moiety rather than the protein core. Therefore, these data suggested that the target cell recognition molecule which is recognized by mAb 36 appears to be a membrane carbohydrate-associated molecule.

Mechanism of target cell recognition by natural killer cells: characterization of a novel triggering molecule restricted to CD3- large granular lymphocytes.

In an attempt to identify a molecule in target recognition by CD3- large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3- LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3- LGL with F(ab')2 fragments resulted in the release of serine esterases and the secretion of interferon gamma. Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti-DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK)-susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function.

Variable efficacy of interferon-alpha treatment on growth of human hepatoma cell lines in vitro.

Three human hepatoma cell lines, PLC/PRF/5, Mahlavu and Sk-Hep 1, two of which contain integrated HBV DNA, were grown in culture and treated with human alpha-IFN for up to 14 days. IFN treatment caused a varying suppression of cell growth of the three hepatoma cell lines. While doubling time and cloning efficiency were significantly reduced for all three hepatoma cell lines tested, 3[H]thymidine incorporation was markedly suppressed, in a dose-dependent fashion, only in treated PLC/PRF/5 cells but not in Sk-Hep 1 and Mahlavu cells. The inhibiting effect of interferon treatment on growth of PLC/PRF/5 cells in vitro was neutralized by antibodies to human IFN. IFN treatment caused a significant suppression of HBsAg and alpha FP secretion by PLC/PRF/5 hepatoma cells. This effect, while constant throughout the observation period for HBsAg, was cumulative for alpha FP secretion. Following discontinuation of treatment, suppression of PLC/PRF/5 hepatoma cell growth was rapidly reversed, and HBsAg and alpha FP secretion returned to their pretreatment levels. These experiments suggest that human alpha-IFN suppresses the growth of some human hepatoma cells in culture but that this effect is dependent on the continuous presence of IFN in the growth medium. Finally, the inhibitory effects of IFN on cell growth differed for the various hepatoma cell lines tested.

Human cell lines secreting lymphokines. II. BCGF production by a T hybridoma and B cell lines.

B cell growth factor (BCGF) is a lymphokine (LK) primarily produced and secreted by activated T cells. This LK induces proliferation of B cells in culture and can maintain continuous growth of human B cells. One of the putative uses of this factor might be the establishment of monoclonal B cell lines which secrete specific antibodies. These antibodies could be used for passive immunization or in vivo immunodiagnostics. As conventional mitogen activation of T cells induces the secretion of many factors, some of them with opposite effects, the approach taken by us and others was to establish monoclonal hybridomas which produce constitutively a single factor. Such a human T-T cell hybridoma (TH-5), has been established and is growing and secreting constitutively BCGF for the tested 18 months. This BCGF induces proliferation of human B-cells without the requirement of B cell preactivation. No secretion of interleukin-2 or gamma-interferon by this hybridoma was detected. Furthermore, no influence of this factor was detected on T cells. Under optimal growth conditions, the generation time of this hybridoma is 16 hours and it reaches a maximum concentration of 3 X 10(6) cells/ml. The hybridoma cells could grow in serum-free-media and secrete BCGF for a limited time. The produced BCGF was found to be stable for the four tested months at -20 degrees C, at least one week at 4 degrees C and several hours at 37 degrees C. Its activity was destroyed at pH 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocompetence in pregnancy: production of interleukin-2 by peripheral blood lymphocytes.

Pregnancy is a natural allograft and the mechanisms for its non-rejection are obscure. Depression of maternal cellular immunity was suggested as a possible explanation. Interleukin-2(IL-2) is a lymphokine release from OKT4+ lymphocyte. This factor has a crucial role in the proliferation and differentiation of T cell subsets, and controls functions associated with immune rejection mechanisms. We therefore examined the ability of lymphocytes from women in the 3 trimesters of pregnancy to produce IL-2 in culture. Mononuclear cells were cultured with PHA for 48 h. The IL-2-containing supernatant was added to and supported the proliferation of an IL-2 dependent T cell line. Proliferation of this line indicated the IL-2 content of the added supernatant. Using this assay, IL-2 production in all 3 trimesters of pregnancy was adequate and comparable to that of lymphocytes from non-pregnant women. These results suggest that the proposed defect in cellular immunity during pregnancy is not mediated by an inability of the lymphocytes to produce IL-2.

EMC virus infection in baboons as a model for studies on antiviral substances.

EMC virus causes a lethal infection in baboon monkeys within 4-8 days following subcutaneous injection with 10(4)-10(8) pfu of virus. The infection is accompanied by viremia, invasion of heart muscle and of brain. Monkeys infected with 10(6) pfu of EMC virus were treated with human leukocyte interferon. The interferon was injected intramuscularly first 0, 0.5, 6 and 24 h post-infection, then twice daily with a dose of 3 X 10(6) units for 5 consecutive days. All the monkeys treated with interferon remained alive and healthy. Animals infected with EMC virus, but not treated with interferon died within 6 days with evidence of myocarditis. The EMC virus-interferon interaction in baboon monkeys seems to provide a useful primate model system for testing the prophylactic and therapeutic antiviral activity of interferons or other antiviral substances.

Interleukin-2 production by cord blood lymphocytes stimulated with mitogen and in the mixed leukocyte culture.

We examined the ability of cord blood lymphocytes to produce interleukin-2 (T cell growth factor) in response to phytohemagglutinin and in the mixed leukocyte culture. Interleukin-2 production was measured by the proliferative response of an interleukin-2-dependent mouse T cell line to the addition of supernatant obtained from cord blood lymphocyte cultures. Using these assays we have shown that cord blood lymphocytes have a normal ability to produce interleukin-2 in both PHA-stimulated cultures and in the mixed leukocyte culture. The normal production of interleukin-2 by cord blood cells indicates, that newborn T lymphocytes are mature, and that a dissociation exists between their normal ability to produce interleukin-2 and their ability to produce other lymphokines, which was reported to be impaired. The reported deficiency of HLA-DR antigen expression on newborn monocytes does not seem to interfere with the production of interleukin-2 in the mixed leukocyte culture.

The fate of [125I]-labeled human leukocyte-derived alpha interferon in the rat.

In order to follow the catabolic fate of interferon in the body, [125I]-labeled human alpha interferon was infused into rats. Interferon activity and TCA-precipitable radioactivity in the blood reached a steady state after 60 min of infusion, while TCA-soluble radioactivity continued to increase during the entire infusion period. A massive accumulation of both active interferon and degradation products of interferon were found in the kidney at the end of the infusion. Most of the interferon activity was found in the mitochondrial-lysosomal fraction, while most of the interferon degradation products were found in the supernatant fraction. Ligation of the kidney's blood vessels resulted in a large increase of interferon activity in the blood. Only a small increase was found in other organs, with no increase at all in radioactive degradation products. Our results suggest the kidney to be the main site of interferon catabolism. Low molecular weight degradation products are excreted from the kidney into the blood and are at least partially secreted in the urine and also taken up by other organs.

Interleukin-2 production and response to exogenous interleukin-2 in a patient with the acquired immune deficiency syndrome (AIDS).

The ability of lymphocytes from a patient suffering from the acquired immune deficiency syndrome (AIDS) to produce interleukin-2 (IL-2) was found to be comparable to that of his healthy sex partner and to that of a normal control. Addition of exogenous IL-2 to lymphocyte cultures did not improve the poor mitogen response to phytohaemagglutinin in this patient. Our data suggest that the underlying defect in this AIDS patient is due to an IL-2 receptor defect.

Treatment of life-threatening viral infections with interferon alpha: pharmacokinetic studies in a clinical trial.

The effectiveness of human leukocyte interferon (IFN alpha) therapy was studied in 15 patients with acute life-threatening viral illnesses. All patients were critically ill, many close to death, when IFN therapy was begun. Included were six patients with acute fulminant hepatitis, four immunosuppressed patients with spreading herpes simplex, three severely ill patients with encephalitis, one case of severe fulminant juvenile laryngeal papillomatosis, and one of postmeasles dermatitis. Twelve of the 15 patients recovered, some dramatically, including 3 of the 6 fulminant hepatitis patients. Pharmacokinetic studies showed defective antiviral IFN responses in most of the patients--in particular, absence of in vivo IFN production. Because the patients were not producing IFN in response to the viral infection, the peripheral blood mononuclear cells were not primed into an antiviral state. Treatment with IFN alpha led to the rapid development of an antiviral state of the cells, which paralleled clinical recovery. In our opinion, IFN is the treatment of choice in acute viral infections, often lifesaving, provided it is given early in the infection before irreversible cell and tissue damage has taken place. Its use is most effective in those seriously ill patients with defective antiviral IFN responses.

Involvement of the kidney in catabolism of human leukocyte interferon.

The metabolic fate of human leukocyte interferon (HuIFN-alpha) was studied after intravenous injection into rats and cynomolgus monkeys. At various intervals the animals were sacrificed and the HuIFN-alpha content determined in serum and various tissues. HuIFN-alpha quickly disappeared from the circulation and was found mainly in the kidneys, in which levels were at least 7- to 10-fold higher than in the liver, spleen, lungs, heart, brain and muscles. No interferon was detected in urine. Subcellular fractionation of kidney revealed that the mitochondrial-lysosomal fraction (15 000 g) had a high HuIFN-alpha content. It was also found that HuIFN-alpha was rapidly inactivated by two types of proteinases found in the lysosomal fractions of rat, monkey and human kidneys, with an optimal pH of 3 to 4. The inactivation was partially inhibited by either pepstatin or leupeptin. Inactivation was totally prevented by a mixture of both inhibitors. Since it is known that interferon is scantily excreted in urine, our findings suggest that the kidney serves as a main site for its degradation.

The kidney is the main site of interferon degradation.

Male Sprague Dawley rats (300 g) were infused by constant infusion into the jugular vein through 3-5 h with human leukocyte-derived interferon (HuIFN-alpha 10(6) units/h, 0.9 ml/h). Blood samples were collected every 20 min through the carotid. A steady-state level of interferon in serum (10(4) U/ml) was reached after 50 min of infusion, indicating fast removal of most of the infused material. Examination of interferon distribution in various tissues at the termination of the infusion revealed that over 85% of active interferon was found in the kidneys. No interferon was found in urine. Ligation of both kidneys resulted (after 2.5 h of infusion) in a ten-fold increase in interferon in serum and a minor increase in other tissues. No interferon was found in the kidneys. Simultaneous infusion of HuIFN-alpha, pepstatin and leupeptin (the well-known inhibitors of lysosomal proteinases) for 2.5 h had no effect on interferon concentration in serum and various tissues except in kidneys where a 5-fold increase was found. Mitochondrial-lysosomal fractions contained 55% of the total kidney interferon. Partial inhibition of proteolytic and HuIFN-alpha degrading activities at that fraction was also observed. These data, together with our previous findings of accumulation of injected interferon in the mitochondrial-lysosomal fraction of rats and monkeys' kidney cells, provide a further evidence for the main role of the kidney in HuIFN-alpha degradation.