A novel herpesvirus in 3 species of pheasants: mountain peacock pheasant (Polyplectron inopinatum), Malayan peacock pheasant (Polyplectron malacense), and Congo peafowl (Afropavo congensis).

The mountain peacock pheasant (Polyplectron inopinatum), the Malayan peacock pheasant (Polyplectron malacense), and the Congo peafowl (Afropavo congensis) are all listed as vulnerable to extinction under the International Union for Conservation of Nature Red List of Threatened Species. Here the authors report fatal infection with a novel herpesvirus in all 3 species of birds. DNA was extracted from the livers of birds with hepatocellular necrosis and intranuclear eosinophilic inclusions consistent with herpesvirus infection. Based on degenerate herpesvirus primers and polymerase chain reaction, 220- and 519-base pair products of the herpes DNA polymerase and DNA terminase genes, respectively, were amplified. Sequence analysis revealed that all birds were likely infected with the same virus. At the nucleotide level, the pheasant herpesvirus had 92% identity with gallid herpesvirus 3 and 77.7% identity with gallid herpesvirus 2. At the amino acid level, the herpes virus had 93.8% identity with gallid herpesvirus 3 and 89.4% identity with gallid herpesvirus 2. These findings indicate that the closest relative to this novel herpesvirus is gallid herpesvirus 3, a nonpathogenic virus used widely in a vaccine against Marek's disease. In situ hybridization using probes specific to the peacock pheasant herpesvirus DNA polymerase revealed strong intranuclear staining in the necrotic liver lesions of an infected Malayan peacock pheasant but no staining in normal liver from an uninfected bird. The phasianid herpesvirus reported here is a novel member of the genus Mardivirus of the subfamily Alphaherpesvirinae and is distinct from other galliform herpesviruses.

Suppressive vaccination with DNA encoding a variable region gene of the T-cell receptor prevents autoimmune encephalomyelitis and activates Th2 immunity.

A variable region gene of the T-cell receptor, V beta 8.2, is rearranged, and its product is expressed on pathogenic T cells that induce experimental autoimmune encephalomyelitis (EAE) in H-2u mice after immunization with myelin basic protein (MBP). Vaccination of these mice with naked DNA encoding V beta 8.2 protected mice from EAE. Analysis of T cells reacting to the pathogenic portion of the MBP molecule indicated that in the vaccinated mice there was a reduction in the Th1 cytokines interleukin-2 (IL-2) and interferon-gama. In parallel, there was an elevation in the production of IL-4, a Th2 cytokine associated with suppression of disease. A novel feature of DNA immunization for autoimmune disease, reversal of the autoimmune response from Th1 to Th2, may make this approach attractive for treatment of Th1-mediated diseases like multiple sclerosis, juvenile diabetes and rheumatoid arthritis.

Idiotypic immunization induces immunity to mutated p53 and tumor rejection.

The p53 molecule might serve as a common tumor-associated antigen, as the tumor suppressor gene p53 is mutated and the p53 protein is often over-expressed in tumor cells. We report that effective immunity to p53 can be induced through an idiotypic network by immunization of mice with a monoclonal antibody (PAb-240) specific for mutated p53, or with a peptide derived from the complementarity determining region (CDR) 3 of the variable domain of the light chain (VL) of this antibody. The immunized mice produced IgG antibodies to p53 and mounted a cytotoxic reaction to a tumor line bearing mutated p53. The idiotypically immunized mice were resistant to challenge with the tumor cells. Thus antibodies to p53 might serve as immunogens for activating resistance to some tumors. At the basic level, these findings indicate that a network of p53 immunity may be organized naturally within the immune system.

Microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis.

Molecular mimicry refers to structural homologies between a self-protein and a microbial protein. A major epitope of myelin basic protein (MBP), p87-99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE). VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex. Peptides from papilloma virus strains containing the motif VHFFK induce EAE. A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE. A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE. T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE. Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87-99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease.

Recovery of visual response of injured adult rat optic nerves treated with transglutaminase.

Failure of axons of the central nervous system in adult mammals to regenerate spontaneously after injury is attributed in part to inhibitory molecules associated with oligodendrocytes. Regeneration of central nervous system axons in fish is correlated with the presence of a transglutaminase. This enzyme dimerizes interleukin-2, and the product is cytotoxic to oligodendrocytes in vitro. Application of this nerve-derived transglutaminase to rat optic nerves, in which the injury had caused the loss of visual evoked potential response to light, promoted the recovery of that response within 6 weeks after injury. Transmission electron microscopy analysis revealed the concomitant appearance of axons in the distal stump of the optic nerve.

Enhancement of the in vitro and in vivo antitumor activities of phosphorylated mitomycin C and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates.

Alkaline phosphatase (AP) was covalently linked to the two antitumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), forming conjugates that could bind to antigen-positive tumor cells. The conjugates were able to convert the prodrugs, mitomycin phosphate (MOP) and etoposide phosphate (EP), into an active mitomycin C derivative, mitomycin alcohol, and etoposide, respectively. MOP and EP were less toxic to cultured cells from the H2981 lung adenocarcinoma than their respective hydrolysis products, mitomycin alcohol and etoposide, by a factor greater than 100, and they were also less toxic in mice. Pretreatment of H2981 cells with L6-AP greatly enhanced the cytotoxic effects of MOP and EP, while 1F5-AP caused no such enhancement. A strong antitumor response was observed in H2981-bearing mice that were treated with L6-AP followed 24 h later by either MOP or a combination of MOP and EP. This response was superior to that of MOP or combinations of MOP and EP given alone.

Macrophage recruitment to acutely injured central nervous system is inhibited by a resident factor: a basis for an immune-brain barrier.

Compared to the peripheral nervous system (PNS), the central nervous system (CNS) of mammals has a poor prospect for regeneration. Accumulating evidence suggests that this is due, in part, to differences in how the immune and nervous systems communicate in response to injury. The macrophage is one of the central cells in this communication with the capacity to respond in a variety of ways depending on the conditions of stimulation. After injury, macrophages enter the CNS much later and in fewer numbers than they do the PNS. It is possible that this late and reduced response is not sufficient to modify the CNS environment to one that is conducive to successful regeneration. In the present study we investigated whether the limited macrophage invasion of injured CNS is due to the presence of an endogenous inhibitory factor that is persistent after injury. Using an in vitro migration assay, we show that rat optic nerve (CNS) is deficient in its ability to attract monocytes as compared to rat sciatic nerve (PNS). We further demonstrate that this deficiency is due to the presence of a soluble inhibitory factor in the CNS. This factor may also cause a subsequent effective difference in those macrophages that are recruited, as is shown by morphological data. The brain-resident factor that inhibits macrophage migration may be the physiological basis of an immune-brain barrier underlying the known phenomenon of immune privilege.

Inflammation after axonal injury has conflicting consequences for recovery of function: rescue of spared axons is impaired but regeneration is supported.

Neural injury leads to tissue damage beyond that caused by the initial lesion, mainly as a result of a chain of autodestructive events triggered by the trauma. These events apparently include the activation of immune-derived cells and their products, as treatment with anti-inflammatory agents, such as corticosteroids, limits the damage and thus improves recovery. On the other hand, immune-derived substances, such as cytokines, are thought to play an important role in post-traumatic axonal regeneration. Thus, the need to reduce inflammation to limit the spread of damage appears to be in conflict with the need to permit inflammation to promote regeneration. Comprehension and resolution of this apparent conflict may lead to the development of treatment protocols aimed at rescuing axons spared by the initial injury, without hampering the potential regeneration of directly and indirectly injured axons. In this study, carried out on rats with crushed optic nerves, daily intraperitoneal injections of dexamethasone commencing prior to the injury significantly attenuated the injury-induced decrease in electrophysiological activity and reduced the area of tissue damage. On the other hand, dexamethasone treatment reduced the permissiveness of the injured nerves to neural adhesion and regrowth in vitro. This latter phenomenon was also observed in injured peripheral nerves. Results are discussed with respect to the possible establishment of an appropriate protocol for corticosteroid treatment of nerve injuries aimed at promoting neuronal rescue without compromising neuronal regeneration.

Accumulation of passively transferred primed T cells independently of their antigen specificity following central nervous system trauma.

The central nervous system (CNS) enjoys a unique relationship with the immune system. Under non-pathological conditions, T cells move through the CNS but do not accumulate there. CNS trauma has been shown to trigger a response to CNS self-antigens such as myelin basic protein (MBP). Here, we examined whether the injured CNS tissue undergoes changes that permit T cell accumulation. We found that injury to CNS white matter, such as the optic nerve, led to a transiently increased accumulation of T cells (between days 3 and 21). In Lewis rats with unilaterally injured optic nerves, systemic administration of passively transferred T cells recognizing either self-antigen (MBP) or non-self-antigen (ovalbumin) resulted in accumulation of the T cells in injured optic nerve, irrespective of their antigenic specificity. The effect of the T cells on the damaged nerve, the lack of selectivity in T cell accumulation and the mechanism underlying non-selective accumulation are discussed.

Linear dimeric interleukin-2 obtained by the use of a defective herpes simplex viral vector: conformation-activity relationship.

An interleukin-2 dimer, produced enzymatically by a nerve-derived transglutaminase in vitro, is cytotoxic to oligodendrocytes, unlike the immune-derived monomeric interleukin-2. The object of this study was to establish a way to produce a dimer of interleukin-2 in quantities, by means of genetic engineering, and to confirm that the structure of the resulting molecule is critical for its function. A defective herpes simplex virus vector was utilized for overproduction of a dimeric interleukin-2. The resulting linear dimer, which is a translational product, differs from the enzymatically produced dimer, which is a posttranslational modification of interleukin-2. The linear dimer, while retaining the known interleukin-2 activity of monomeric interleukin-2 with respect to mitogenicity on T cells, was not cytotoxic to oligodendrocytes. This finding suggests that the lack of cytotoxicity of the linear dimeric interleukin-2 is not caused by a loss of activity during its preparation but is related to its conformational structure, which evidently does not meet the requirements for cytotoxicity. This study opens the way to the design at the transcriptional level of modified proteins and their efficient production, provided that the new transcript encodes for the desired modification in the protein at the appropriate sites.

Soluble factor(s) produced in injured fish optic nerve regulate the postinjury number of oligodendrocytes: possible role of macrophages.

Mammalian central nervous system (CNS) axons are virtually incapable of regenerating after injury. However, CNS neurons of lower vertebrates, such as fish and amphibians, are endowed with a high regenerative capacity. Lately, the glial cells have been credited with the regenerative ability of any specific CNS. We have previously demonstrated that many oligodendrocytes are recovered in cultures of injured rat optic nerve, while only a few oligodendrocytes are recovered from injured fish optic nerve in culture. We further demonstrated that medium conditioned by regenerating fish optic nerves (CM), which has been shown to cause axonal elongation in injured rabbit optic nerves, causes a decrease in the number of oligodendrocytes in rat glial cultures. In the present study, we demonstrate that soluble factors in the CM are capable of reducing the number of fish oligodendrocytes in fish optic nerve cultures. In addition, an inverse relationship was found between the number of macrophages and the number of oligodendrocytes. These results thus suggest that macrophages and/or activated resident microglial cells are directly or indirectly responsible for the presence of these soluble factor(s) that regulate the postinjury number of oligodendrocytes in the fish optic nerves.

Monoclonal antibody L6-daunomycin conjugates constructed to release free drug at the lower pH of tumor tissue.

Measurements in cancer patients showed that the pH of tumors averages 0.8 unit lower than that of the surrounding normal tissues, confirming published work. Based on this, the anti-carcinoma monoclonal antibody (mAb) L6 was used to prepare immunoconjugates with daunomycin (DM), the drug being released at the acidic pH of the tumor. A direct linking of the aconitic derivative of DM (AcoDM) to mAb L6 led to conjugates that either had a low drug/antibody ratio (less than 5:1) or precipitated in vitro. In order to increase the drug load and avoid precipitation, several biopolymers were tested as spacers between the drug and the L6. To attach the polymer derivative to the mAb, the former was maleimidized and the mAb was thiolated. The AcoM/mAb ratio obtained was 20, and the mAb retained its highly specific binding to tumor cells. At pH 6 the AcoDM-L6 conjugate was toxic to cultured C-3347 carcinoma cells with an inhibitory concentration (IC50) of 5 micrograms/ml. The conjugate was less effective than the free DM with an IC50 of 0.2 micrograms/ml. The L6 alone was not toxic. At a tumor pH of 6.5, 15% of the AcoDM was released. The amount of released drug reached a maximum 24-48 h after exposure to the acidic medium. In vivo localization studies demonstrated a similar tumor uptake of the conjugate and mAb L6 with 18% of the injected dose/g tumor and a maximum uptake in tumor 48 h after injection. Our data indicate that it is possible to construct conjugates based on a pH-sensitive linker that can be targeted successfully to a tumor with release of a portion of the drug at the tumor site, but testing is needed to establish whether such release has anti-tumor activity in vivo and offers an advantage over treatment with unconjugated drug.

Transplantation of activated macrophages overcomes central nervous system regrowth failure.

Macrophages have long been known to play a key role in the healing processes of tissues that regenerate after injury; however, the nature of their involvement in healing of the injured central nervous system (CNS) is still a subject of controversy. Here we show that the absence of regrowth in transected rat optic nerve (which, like all other CNS nerves in mammals, cannot regenerate after injury) can be overcome by local transplantation of macrophages preincubated ex vivo with segments of a nerve (e.g., sciatic nerve) that can regenerate after injury. The observed effect of the transplanted macrophages was found to be an outcome of their stimulated activity, as indicated by phagocytosis. Thus, macrophage phagocytic activity was stimulated by their preincubation with sciatic nerve segments but inhibited by their preincubation with optic nerve segments. We conclude that the inability of nerves of the mammalian CNS to regenerate is related to the failure of their macrophages recruited after injury to acquire growth-supportive activity. We attribute this failure to the presence of a CNS resident macrophage inhibitory activity, which may be the biochemical basis underlying the immune privilege of the CNS. The transplantation of suitably activated macrophages into injured nerves may overcome multiple malfunctioning aspects of the CNS response to trauma, and thus may be developed into a novel, practical, and multipotent therapy for CNS injuries.

Antibody-penicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide.

The two monoclonal antibodies (mAb), L6 (anti-carcinoma), and 1F5 [anti-(B-cell-lymphoma)], were chemically linked to the enzyme penicillin-V amidase (PVA), which hydrolyzes phenoxyacetamides, to explore the potential of using mAb-enzyme conjugates for the localization of chemotherapeutic drugs at tumor cells. The phenoxyacetamide derivatives of doxorubicin and melphalan were prepared, yielding the less toxic amides, doxorubicin-N-p-hydroxyphenoxyacetamide (DPO) and melphalan-N-p-hydroxyphenoxyacetamide (MelPO). These were hydrolyzed by PVA to doxorubicin and melphalan respectively. In vitro studies with the L6-positive lung carcinoma cell line, H2981, and the 1F5-positive B-cell lymphoma line, Daudi, showed that DPO was 80-fold less toxic to H2981 cells and 20-fold less toxic to Daudi cells than doxorubicin, and its toxicity was substantially increased when the H2981 cells were pretreated with L6-PVA or the Daudi cells were pretreated with 1F5-PVA. The cytotoxic effect was antigen-specific, since only the binding mAb-enzyme conjugate increased the cytotoxicity of the prodrug. MelPO was more than 1000-fold less toxic than melphalan to H2981 cells and more than 100-fold less toxic than melphalan to Daudi cells. Pretreatment with the mAb-PVA conjugates did not enhance the toxicity of MelPO in either cell line, because PVA hydrolyzes the phenoxyacetamide bond of MelPO too slowly to generate a toxic level of melphalan.

Identification of an interleukin 2-like substance as a factor cytotoxic to oligodendrocytes and associated with central nervous system regeneration.

Axons of the central nervous system in adult mammals do not regenerate spontaneously after injury, partly because of the presence of oligodendrocytes that inhibit axonal growth. This is not the case in lower vertebrates (e.g., in fish), where regeneration of the optic nerve does occur spontaneously and has been correlated with the presence of factors cytotoxic to oligodendrocytes. The present study provides evidence that the substance originating from the fish optic nerves, which is cytotoxic to oligodendrocytes, is an interleukin 2-like substance.

Combination of gene delivery and DNA vaccination to protect from and reverse Th1 autoimmune disease via deviation to the Th2 pathway.

Using a combination of local gene delivery and tolerizing DNA vaccination, we demonstrate that codelivery of the interleukin-4 (IL-4) gene and a DNA vaccine encoding the self-peptide proteolipid protein 139-151 (PLP139-151) provides protective immunity against experimental autoimmune encephalomyelitis (EAE). We provide evidence for a mechanism whereby IL-4 expressed from the naked DNA is secreted and acts locally on autoreactive T cells via activation of STAT6 to shift their cytokine profile to T helper 2. We also show that DNA vaccines can be used to reverse established EAE by covaccination with the genes for myelin oligodendrocyte glycoprotein and IL-4. This treatment strategy combines the antigen-specific effects of DNA vaccination and the beneficial effects of local gene delivery.

Immunomodulation of experimental autoimmune encephalomyelitis with ordered peptides based on MHC-TCR binding motifs.

T cell-mediated destruction of the myelin sheath causes inflammatory damage of the CNS in multiple sclerosis (MS). The major T and B cell responses in MS patients who are HLA-DR2 (about two-thirds of MS patients) react to a region between residues 84 and 103 of myelin basic protein (1 ). The crystal structure of HLA-DR2 complexed with myelin basic protein(84-102) confirmed that Lys(91) is the major TCR contact site, whereas Phe(90) is a major anchor to MHC and binds the hydrophobic P4 pocket (2 ). We have tested peptides containing repetitive 4-aa sequences designed to bind critical MHC pockets and to interfere with T cell activation. One such sequence, EYYKEYYKEYYK, ameliorates experimental autoimmune encephalomyelitis in Lewis rats, an animal model of MS.

Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate.

Two anti-tumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), were covalently linked to alkaline phosphatase (AP), forming conjugates that could bind to the surface of antigen-positive tumor cells. The conjugates were capable of converting a relatively noncytotoxic prodrug, etoposide phosphate (EP), into etoposide--a drug with significant antitumor activity. In vitro studies with a human colon carcinoma cell line, H3347, demonstrated that while EP was less toxic than etoposide by a factor of greater than 100, it was equally toxic when the cells were pretreated with L6-AP, a conjugate that bound to the surface of H3347 cells. The L6-AP conjugate localized in H3347 tumor xenografts in nude mice and histological evaluation indicated that the targeted enzyme (AP) was distributed throughout the tumor mass. A strong antitumor response was observed in H3347-bearing mice that were treated with L6-AP followed 18-24 hr later by EP. This response, which included the rejection of established tumors, was superior to that of EP (P less than 0.005) or etoposide (P less than 0.001) given alone. The IF5-AP conjugate did not bind to H3347 cells and did not enhance the toxicity of EP on these cells in vitro. In addition, IF5-AP did not localize to H3347 tumors in nude mice and did not demonstrate enhanced antitumor activity in combination with the prodrug.

Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis.

Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.