Search

Acute mesenteric ischemia (AMI) is caused by considerable intestinal injury, which is associated with intestinal ischemia followed by reperfusion. To elucidate the mechanisms of ischemia/reperfusion injuries, a C5a inhibitory peptide termed AcPepA was used to examine the role of C5a anaphylatoxin, induction of inflammatory cells, and cell proliferation of the intestinal epithelial cells in an experimental AMI model. In this rat model, the superior mesenteric artery was occluded and subsequently reperfused (Induce-I/R). Other groups were treated with AcPepA before ischemia or reperfusion. Induce-I/R induced injuries in the intestine and AcPepA significantly decreased the proportion of severely injured villi. Induce-I/R induced secondary receptor for C5a-positive polymorphonuclear leukocytes in the vessels and CD204-positive macrophages near the injured site; this was correlated with hypoxia-induced factor 1-alpha-positive cells. Induction of these inflammatory cells was attenuated by AcPepA. In addition, AcPepA increased proliferation of epithelial cells in the villi, possibly preventing further damage. Therefore, Induce-I/R activates C5a followed by the accumulation of polymorphonuclear leukocyte and hypoxia-induced factor 1-alpha-producing macrophages, leading to villus injury. AcPepA, a C5a inhibitory peptide, blocks the deleterious effects of C5a, indicating it has a therapeutic effect on the inflammatory consequences of experimental AMI.

Performance status (PS) frequently improves occurs in cancer patients who have been infused with their own lymphokine-activated killer T cells (LAK-T). In the present study, a culture supernatant of LAK-T (LAK-T sup) administered to 8-week-old rats caused neurogenesis as evidenced by increased 5-ethynyl-2'-deoxyuridine staining of brain tissues. Intravenous injection of granulocyte-macrophage colony stimulating factor (GM-CSF), a major cytokine in LAK-T sup, had a similar effect. Furthermore, LAK-T sup induced Ca(++) increase in rat hippocampal brain slices that was detected in neuronal cells by emission of Fluo-8 NW at 520 nm. The same effect was observed with an rGM-CSF solution. GM-CSF may activate neuronal cells by stimulating the glial cells that surround and attach to them. If so, GM-CSF and LAK-T sup may improve the motor neurons of patients with amyotrophic lateral sclerosis. The neurogenerative effect of GM-CSF in LAK-T sup may also help improve brain function in aged adults including those with dementia such as Alzheimer's disease.

Amino acids 37 to 53 (RAARISLGPRCIKAFTE) of C5a anaphylatoxin form an essential region for C5a function. To target this sequence, we generated a complementary peptide (ASGAPAPGPAGPLRPMF) designated PepA which has a potent inhibitory effect on C5a activity. By introducing an acetyl group at the N-terminal alanine of PepA, an acetylated form was generated which was designated AcPepA. The acetylation resulted in increased inhibition of C5a stimulation of neutrophils as determined by Ca influx. Furthermore, AcPepA partially inhibited the lethal shock induced in mice by intravenous administration of Candida albicans water-soluble mannoprotein-beta-glucan complex. In addition, local skin inflammation in rats caused by an anti-Crry monoclonal antibody was suppressed when AcPepA and the antibody were injected together, while PepA had little inhibitory capacity. The potent inhibitory capacity of AcPepA was also confirmed by a skin reaction of guinea pigs inoculated with recombinant human C5a together with AcPepA.

The trans-chromosome (TC) mouse that we used harbors human chromosomes 2, 14 and/or 22, and has undergone knock-out of its endogeneous genes coding for mu-and kappa-chains of immunoglobulin. One of these TC mice was immunized with HIV-1-infected U937 cells, and spleen cells from the immunized animal were fused with the mouse myeloma cell line to generate hybridoma cells. We selected hybridomas that produce human IgM antibodies (Abs) reactive with HIV-1-infected MOLT4 cells but not with uninfected MOLT4 cells. Two hybridoma cell lines were established termed 9F11 and 2G9. Although 0.4 mug/ml of 9F11 was able to induce complement-mediated cytolysis of the infected cells in the presence of fresh human serum, 2G9 could not. There was no difference between the two monoclonal Abs in the base sequences of cDNAs coding for the constant regions of mu-and kappa-chains. Therefore, we speculate that the ability to activate complement on homologous cell membranes might reflect the structural presentation of antigenic molecules, which could facilitate the binding of an IgM Ab to multiple binding sites resulting in escape from restriction by species-specific inhibitors of complement such as DAF (CD55) and CD59. On the other hand, 2G9 induced apoptosis of HIV-1-infected cells, including latently infected OM10.1 cells, although the Ag for 2G9 remains to be identified. Since both of the Abs had reduced reactivity toward HIV-1-infected MOLT4 cells following cultivation in the presence of tunicamycin, the responsible antigens would involve a sugar moiety.

Procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is present in plasma and can be activated to carboxypeptidase R (CPR) by trypsin-like enzymes such as thrombin and plasmin. CPR has the carboxypeptidase B-like activity that can inactivate the inflammatory peptides such as C5a by removing the C-terminal arginine and can interfere with fibrinolysis by removing C-terminal lysine residue of fibrin. In the present study, we conducted to produce monoclonal antibodies (mAbs) by using spleen cells from proCPR-deficient mice immunized by partially purified mouse proCPR. The mAbs obtained were IgM isotype and reacted with proCPR and interfered with activation of proCPR to CPR by thrombin-thrombomodulin complex. Some BALB/c mice implanted with the hybridoma died in 7 days, and intravenous injection of the mAb to BALB/c mice induced transient elevation of GOT and GPT in plasma although injection to the deficient mice did not. Furthermore, the histological features showed the focally lesions in liver tissue of BALB/c mice injected with the mAb. Since liver is the major site of proCPR synthesis, IgM mAb to proCPR should have induced local inflammation at the side resulting in induction of hepatitis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) was reported as an anaphylatoxin-inactivating enzyme generated by proteolytic cleavage of its zymogen, and is the same enzyme as that first designated by our group as procarboxypeptidase R (proCPR). Its level in plasma appears to influence vascular disease. In addition, TAFI activity is strongly influenced by genetic polymorphism, especially at amino acids 147 and 325. We investigated whether these TAFI polymorphisms would act as a risk factor for cerebral infarction (CI) by examining 253 samples in which the diagnosis was cliniconeuropathologically confirmed. We found little that was statistically significant in terms of these polymorphisms among patients with no vascular problems or in a population-based control group. In the present study of an elderly Japanese group, our samples revealed a lower percentage of the Ile allele at Thr/Ile-325 compared with western counterparts. Although patients with severe infarcts had a lower percentage of the Ile allele (10%) at amino acid position 325 compared with the slightly and moderately affected patients and the population-based control group (15-18%), no statistical significance was found. None of our results showed any statistical correlation between TAFI polymorphisms and CI.

Alpha-fetoprotein (AFP) expression is observed in embryonic tissues and, the expression of this protein is absent in normal adult tissues. The re-elevation of serum AFP strongly suggests generation of a malignant tumor in an adult. We demonstrated here that AFP-producing gastric cancer (AFP-gastric cancer) could be treated by a combination therapy with a low dose of Mitomycin-C (MMC) and lymphokineactivated killer T (LAK-T) cells. Treatment with MMC of AFP-gastric cancer cells enhanced their susceptibility to LAK-T cells and induced ATBF1 gene expression. We revealed here a novel signal pathway for regulation of the cell cycle of AFP-gastric cancer cells through ATBF1, which enhances the promoter activity of the p21 (Waf1/Cip1) gene. Immunoprecipitation revealed the direct interaction between ATBF1 and p53. Overexpressed ATBF1 stimulated p21 (Waf1/Cip1) promoter activity up to 4-fold compared with basal activity. The expression level of ATBF1 mRNA was doubled by MMC (0.05 microg/ml) treatment. The MMC treatment and ATBF1 overexpression synergistically activated the p21 (Waf1/Cip1) promoter activity in a dose-dependent manner up to 7-fold compared with basal activity.

Carboxypeptidase R (EC 3.4.17.20; CPR) is an unstable basic carboxypeptidase found in fresh serum in addition to carboxypeptidase N (CPN) which is a stable enzyme. CPR in fresh serum is generated from its zymogen (proCPR) during coagulation by trypsin-like enzymes such as thrombin and thrombin/thrombomodulin complexes. Since removal of the C-terminal arginine abrogates the anaphylatoxin activity of C3a and C5a, CPR and CPN are regarded as anaphylatoxin inactivators. We report here that the culture supernatant of activated human neutrophils converts proCPR to CPR. Addition of an elastase specific inhibitor, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (MSAAPVCK) to the supernatant of stimulated neutrophils completely inhibited activation of proCPR. On the other hand, a thrombin specific inhibitor, p-Nitrophenyl-p'-amidinophenyl-methanesulfonate hydrochloride (pNP-pAPMS) inhibited only 16% of proCPR activation by the neutrophil supernatant. Furthermore, purified elastase converted proCPR to CPR. Therefore, elastase can activate proCPR directly, or indirectly through activation of some proteases, which have been contaminating in reagents. Release of CPR generating enzymes from neutrophils should play an important role in regulation of excess inflammation.

We designed a new computer program (MIMETIC), which generates a series of peptides for interaction with a target peptide sequence. The genetic algorithm employed ranks the sequences obtained from one generation to the next by "goodness of fit" to the target. MIMETIC designed recognition peptides to various regions of HIV-1 reverse transcriptase. Among ten peptide candidates synthesized, three inhibited reverse transcription in vitro. TLMA2993 and PSTW1594 both targeted the connection domain of reverse transcriptase and ESLA2340 targeted the thumb domain.

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b-9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b-9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59(-) human B-cells, which formed non-selective ion channels with a total conductance of 360 +/- 24 pS as measured at the beginning of the steady-state phase of the inward currents. C5b-8 and small-size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59(+) human B-cells in spite of small-size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59(-) cells inhibited the serum-evoked inward current. The ion channels formed by the small-size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b-8 as well as that of small-size C5b-9. These results offer an explanation as to why CD59-expressing cells are not leaky in spite of a buildup of homologous C5b-8 and small-size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.

Carboxypeptidase R (CPR) exists in precursor form (proCPR) in plasma in contrast to carboxypeptidase N (CPN), which is present in the active state. CPR plays two important roles, one of which appears to be the control of the inflammatory response by inactivation of anaphylatoxins such as complement-derived C3a and C5a. Therefore, an increase in CPR activity may facilitate rapid inactivation of these inflammatory mediators generated at the site of bacterial infection. Upregulation of proCPR expression during the inflammatory response initiated for instance by endotoxin (lipopolysaccharide) should play a role in suppressing hyper-reactivity as seen in septic shock. CPR also functions as an inhibitor of fibrinolysis, where its ability to prevent binding of plasminogen to lysine residues on fibrin clots significantly lengthens tissue plasminogen activator (tPA)-induced fibrinolysis time. Therefore, upregulation of proCPR production during the inflammatory response may exacerbate thrombosis contributing to the development of disseminated intravascular coagulation as well as other conditions involving thrombosis. Co-administration of tPA and a specific inhibitor of CPR, such as potato carboxypeptidase inhibitor, which does not affect CPN, may be useful in thrombolytic therapy.

Carboxypeptidase R (EC 3.4.17.20) (CPR) and carboxypeptidase N (EC 3.4.17.3) (CPN) cleave carboxy-terminal arginine or lysine residues from biologically active peptides such as kinins or anaphylatoxins in the circulation thereby regulating their activities. Although CPN is present in a stable active form in plasma, CPR is generated from proCPR, a plasma zymogen, by proteolytic enzymes such as thrombin, thrombin-thrombomodulin complex and plasmin. We have isolated rat proCPR and CPN cDNA clones which can induce enzymatic activities in culture supernatants of the transfected cells. mRNA of proCPR was detected only in rat liver by Northern hybridization and showed hepatocyte-specific expression. Expression of proCPR mRNA was enhanced following LPS injection, indicating that proCPR production is increased under inflammatory conditions.

HIV-1 infection induces the expression of high level of GM2 ganglioside on infected cells and IgM antibody (Ab) against GM2 can cause complement (C)-mediated cytolysis of HIV-1-infected cells. Since GM2 is immunogenic in human, we proposed that an anti-GM2 IgM Ab may be produced by some HIV-1-infected patients and the titer of this Ab might provide some insight into the progress of the disease. On this premise, the amount of IgM Ab against GM2 was determined in 124 HIV-1-infected patients and 111 seronegative donors. As expected, the anti-GM2 IgM Ab titers of the patients was significantly higher than that of the seronegative donors while the total IgM levels remained unchanged. In addition, we determined the CD4+ cell count and the HIV-RNA load in the HIV-1-infected patients. The results showed a positive correlation between the anti-GM2 IgM Ab titer and CD4+ cell count but a negative correlation between the anti-GM2 IgM Ab titer and HIV-RNA load. These suggest that anti-GM2 IgM Ab induced and/or enhanced by HIV-1 infection causes C-mediated cytolysis of HIV-1-infected cells in vivo to a certain extent, and may help lower the plateau level of the HIV-RNA load. Therefore, the amount of IgM Ab against GM2 may be related to the prognosis of HIV-1 infected patients.

Summary Decay accelerating factor (DAF, CD55) expressed in human reproductive organs and gametes is thought to play a pivotal role in protection against autologous complement activation in the genital tract. To further investigate the role of DAF in reproduction, we analysed DAF distribution in reproductive organs using guinea-pigs that express multiple DAF isoforms. In males, significant staining was observed in testis on the elongated spermatids and spermatozoa. Levels of DAF mRNA with a shorter 3′ untranslated region were significantly enhanced in testis from 9 weeks of age, indicating the presence of DAF mRNA and protein synthesis of spermatozoa DAF in late haploid germ cells. Epididymal spermatozoa appeared to express DAF on the inner acrosomal membrane as well as over their entire surface. Significant DAF expression was also observed on the epithelium of seminal vesicles from 4 weeks of age, with no increase thereafter in the mRNA. C3 mRNA was not detected in this tissue. In females, DAF was detected on the plasma membranes of oocytes through follicle development and on the apical region of uterine epithelium, although the levels of DAF mRNA in these tissues were low. In addition, DAF was selectively expressed on the apical region of ciliated oviductal epithelial cells. The apical region of the ciliated cells comprising the efferent ductule epithelium was also stained significantly, even at 12 days of age, while other epididymal epithelial cells were hardly stained at any age, suggesting that DAF is constitutively expressed on cilia.

Antisense amino acids are amino acids which can be translated from the corresponding anti-codons of a sense amino acid. Antisense peptides encoded by the noncoding DNA strand have a tendency to interact with each other. We have demonstrated that antisense peptide sequences are present intramolecularly, and these may contribute to the folding and maintenance of the tertiary structure of a protein. T20 is a synthetic peptide with an amino acid sequence in the gp41 of HIV-1 and has been demonstrated to be a potent inhibitor of HIV-1 infection. We searched for intramolecular peptide sequences which are antisense to portions of T20. A synthetic peptide (TA-1L) consisting of amino acids 84 to 97 of gp160, which contains an antisense peptide sequence (TA-1) to T20, was shown to inhibit HIV-1(IIIB) infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. The TA-1L site, which exists in the C1 domain of gp160, is highly homologous among strains of HIV-1, especially at TA-1 and in the amino acids flanking the C terminus. Although the TA-1 sites of 18 out of 30 HIV-1 strains were antisense to the T20 region, those of the remaining 12 strains, including HIV-1(MN), were not. However, TA-1L inhibited infection by HIV-1(MN), which has no antisense peptide in T20 corresponding to TA-1, although the inhibitory effect was weaker. TA-1L may thus also interfere with the gp160 interaction with CD4, which has an antisense sequence to TA-1.

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay-accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI-DAF) and a transmembrane form (TM-DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI-DAF or TM-DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI-DAF transfectant cells and established seven unique mAbs (RIKO-1-7). Western blotting analysis using RIKO-3, which reacts with both GPI-DAF and TM-DAF, and RIKO-4, which is an anti-GPI-DAF specific mAb, indicated that GPI-DAF was expressed on erythrocytes, spleen and testis, and that TM-DAF was expressed only in testis.

There are two types of carboxypeptidases present in human blood, carboxypeptidase N (CPN) and arginine carboxypeptidase (CPR). CPR is generated during coagulation from a precursor (proCPR) which can be converted to the active form by trypsin in vitro. Since it is difficult to distinguish the two types of carboxypeptidases in human blood by the measurement of enzyme activity, we established a quantitative sandwich ELISA by which CPR can be quantitated. The amount of CPR in plasma, fresh serum and heated serum were essentially the same. Therefore the ELISA assay does not distinguish proCPR, activated CPR and inactivated CPR. With the ELISA method, CPR was quantitated in plasma from fifty patients with rheumatoid arthritis and eleven patients with severe hepatitis as well as healthy individuals. The amount of CPR in plasma obtained from patients with rheumatoid arthritis was not found to be lower than that of normal subjects. Furthermore, the patients who suffered severe hepatitis and had very low levels of CPR-total were fatal. This suggests that a decrease of CPR level might be a good indication of a patient's prognosis to death by hepatitis.

The presence of C5b-9 complexes, some complement regulators, and abundant cytokines in atherosclerotic lesions has been reported. However, it is unclear whether these complement-associated proteins are produced by vascular smooth muscle cells (SMCs) and how they are influenced by the cytokines. In the present study, we demonstrated, by the reverse transcription-polymerase chain reaction method, the mRNA expression of complement components (C3, C4, and C5) and membrane regulators (decay-accelerating factor, membrane cofactor protein, Crry, and CD59) in cultured SMCs derived from the rat carotid artery. The expression of C9 mRNA was also induced upon stimulation by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and/or lipopolysaccharide (LPS). Northern blot analysis showed that the mRNA expression of C3, C4, DAF and Crry was up-regulated, but that of CD59 was down-regulated by IFN-gamma, TNF-alpha and/or LPS alone or by synergy. The increase of C3 mRNA by TNF-alpha or LPS and that of C4 mRNA by IFN-gamma was induced in a dose-dependent manner. The results indicate that the arterial SMCs of rat have the ability to produce complement components and regulators, which is affected by cytokines and/or LPS. Since atherosclerosis is characterized by the intimal proliferation of SMCs, the complement system including its regulators may be involved in the pathogenesis of the disease.

1 This study set out to examine the endothelin receptor subtypes mediating vasoconstriction in the rat renal arcuate artery. This was done in isolated vessels 120–200 μm in diameter, incubated with a selective agonist and the novel ‘antisense’ peptide to part of the human endothelinA receptor. 2 Groups of vessels (n=6) were incubated with increasing concentrations of endothelin-1 (ET-1), from 1 to 100 nM, which caused a 65% maximal contraction at the highest dose with an pEC50 of 8.16±0.11 M. By contrast, in six other vessels sarafotoxin 6c over the same dose range gave a minimal contraction (around 5% of maximum). 3 Preincubation of six vessels with the antisense peptide ETR p1/f1 at 1 μM had no effect on the ET-1 induced vasoconstriction, in terms of displacement of the concentration-response curve or the maximal tension achieved by the agonist. In the six vessels exposed to 4 μM ETR p1/f1, there was a significant shift of the concentration-response curve and a lower pEC50 at 7.78±0.09 M (P

Damage to autologous tissue by complement is limited by several widely distributed membrane-associated glycoproteins which restrict the action of the complement in homologous species. These include decay accelerating factor (DAF), membrane cofactor protein (MCP) and 20 kDa homologous restriction factor (HRF20,CD59). Using immunohistochemical techniques, we examined the localization of these proteins in the central nervous system (CNS) and peripheral nervous system (PNS) using non-neurological human nervous tissue since some complement components have been demonstrated to be synthesized in the CNS. There was no evidence of parenchymal staining by anti-DAF or anti-MCP antibodies in either type of tissue except for the staining of the endothelium in capillaries. On the other hand, anti-HRF20 antibody clearly stained myelinated axons in the CNS as well as Schwann cells in the PNS. In addition, we detected positive staining by anti-DAF antibody in the PNS of a Paroxysmal nocturnal hemoglobinuria (PNH) patient who is genetically deficient in HRF20.

With the aim of developing an effective immunotherapy for malignant glioma, glioma cells were incubated with tumor necrosis factor-alpha (TNF-alpha) to increase their susceptibility to lysis by lymphokine-activated killer (LAK) cells. Treatment with exogenous TNF-alpha induced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of glioma cells. In addition, the cytolytic activity of LAK cells toward exogenous TNF-alpha treated glioma cells was significantly greater than LAK cell activity toward untreated glioma cells. This increase in cytolytic activity was blocked by anti-ICAM-1 monoclonal antibodies (MAb). Furthermore, co-treatment with a bifunctional antibody (BFA) composed of anti-CD3 (UCHT1) and antiglioma (G-22) antibodies synergistically increased the cytolytic activity of LAK cells towards TNF-alpha-treated glioma cells. These results indicate that a combination of exogenous TNF-alpha and anti-CD3/antiglioma BFA may provide an effective modified adoptive immunotherapy for patients with malignant glioma.

512 antigen, rat complement regulatory factor, has been reported to possess the same structure as Crry/p65, mouse complement regulatory protein, which functions like decay-accelerating factor (DAF) and/or membrane cofactor protein (MCP). This study was designed to evaluate the Nagoya, Japan protective effect on guinea pig cells transfected with 512 cDNA from rat complement mediated lysis. 512 antigen was expressed on rat cells alone, not on guinea pig, pig, or human cells. Guinea pig cells transfected with 512 cDNA were protected from rat complement mediated injury through the inhibition of C3 activation. The magnitude of injury was inversely proportional to the intensity of 512 antigen expression, although the protective effect was not always species-specific. These results demonstrate that the genetic engineering to guinea pig cells with 512 cDNA is effective for the inhibition of rat complement activation.

PROBLEM: We have previously demonstrated that human serum can suppress the proliferative response in autologous mixed lymphocyte reaction (AMLR) in which phytohemagglutinin (PHA)-activated T lymphoblasts act as stimulators (T-TPHA AMLR). The aim of the present work was to determine whether pregnancy serum (PS) possesses an inhibitory capacity similar or different in magnitude. METHODS: Sixteen PS were added to T-TPHA AMLR cultures and the proliferative response was compared with that in the presence of human serum. The effect of PS on the IL-2 dependent proliferation of PHA-activated T Lymphoblasts was examined as well. RESULTS: PS induced a significantly more pronounced suppression of T-TPHA AMLR than human serum (P < 0.05). One PS tested was not inhibitory but rather stimulatory. This PS was obtained from a woman who subsequently had IUGR. The inhibition is related to the existence of a serum inhibitory factor and not to the lack of a serum supporting factor. PS inhibited not only T-TPHA AMLR of the same woman but also T-TPHA AMLR of other individuals as well, implying that the inhibitor is a non-MHC restricted factor. IL-2 dependent cell proliferation was not inhibited by PS, implying that the inhibitor detected in T-TPHA AMLR is not a general cell proliferation inhibitor. CONCLUSIONS: These results suggest that a non-MHC restricted inhibitory factor present in PS may play an important biological role in regulating local immune responses in the fetal-placental unit mediated by autoreactive T cells restricted to autologous activated T lymphocytes.

Hyperacute rejection (from pig to human) is thought to result from activation of complement initiated by the binding of host natural antibodies to α-galactosyl (α-Gal) epitopes of donor endothelial cells. However, α-Gal epitope shares a common precursor with H antigen in humans. This means that H antigens as well as α-Gal epitopes are synthesized in a competitive manner by different enzymes. We thought that it would be possible to convert α-Gal epitopes into H antigens by introducing cDNA of α(1,2)-fucosyltransferase (α1–2FT) into porcine cells, and so, pig embryos were microinjected with αl-2FT cDNA. Transgenic pigs that carried α1–2FT were thus established. Cytotoxicity of fibrocytes derived from skin of transgenic pig was measured by 51Cr release assay, which showed that H antigen-expressing cells were significantly resistant to a challenge with human sera. These experiments indicate that our method provides a new strategy which contributes to a successful discordant xenotransplantation.

It has been known that xenogeneic cells transfected with regulator of complement activation (RCA) molecules such as DAF (CD55), MCP (CD46) and HRF20 (CD59) resist the lysis by the human complement. However, it has not been demonstrated that the induction of double RCA molecules is more effective than that of single RCA molecule to inhibit the human complement attack. In this study, the authors compared the effect on the protection from complement-mediated lysis between single transfection and double transfection of DAF and HRF20 cDNA to bovine aortic endothelial cells (BAEC) using retro viral vector. BAEC transfected with DAF or HRF20 cDNA resisted the lysis by human serum in parallel with the intensity of antigen expression, whereas BAEC transfected with both DAF and HRF20 cDNA resisted the lysis by human serum with anti-BAEC antibody, not human serum alone, more effectively than those with DAF cDNA alone. We conclude that the xenogeneic cells doubly transfected with both DAF and HRF20 cDNA are protected from complement mediated lysis more effectively than those singly transfected with DAF cDNA alone.

HRF20 (CD59) is a membrane glycoprotein which protects cells from the membrane attack reaction of homologous complement. A patient who is completely deficient in HRF20 expression and is suffering from paroxysmal nocturnal hemoglobinuria (PNH) was studied. His parents are cousins and both have decreased HRF20 expression, suggesting that the deficiency is genetic. We established a cultured cell line (NCU1) which is HRF20 deficient from the patient's lymphocytes by Epstein-Barr-virus (EBV) infection. Northern blot analysis revealed HRF20 mRNA signals, indicating that HRF20 mRNA were transcribed. HRF20 cDNA was amplified by the polymerase chain reaction (PCR) method. Sequencing of the cDNA from the NCU1 showed two single-base deletions at amino acid 16 and 96 from the N terminus of the mature protein. Deletion in the genomic DNA of peripheral blood lymphocytes was confirmed by the DNA sequence of an HRF20 open reading frame containing amino acid 16. Furthermore, the patient's parents and sister possessed both intact and deleted genomic HRF20 DNA while his brother's DNA was intact. These findings demonstrate that the HRF20 deficiency was genomic in origin, and that complete deletion was brought about by a homozygous abnormality in the HRF20 gene. The base deletion caused a codon frame shift resulting in failure to produce intact HRF20 protein in the patient.

The 20-kDa homologous restriction factor (HRF20, CD59) is a phosphatidyl inositol-anchored membrane glycoprotein that inhibits the formation of human complement membrane attack complexes. The cDNA of HRF20 was transfected into Chinese hamster ovary (CHO) cells resulting in expression of human HRF20 protein on the cell surface anchored via glycosylphosphatidyl inositol. The transfected CHO cells were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rat and guinea pig complement. Therefore, species specificity between HRF20 and complement is maintained in HRF20 generated on the CHO cells following transfection with HRF20 cDNA.

With the aim of developing an effective cancer immunotherapy for common epithelial cancer, a new class of bifunctional antibody (BFA) was developed; one arm of this BFA recognized c-erbB-2 gene product, and the other arm recognized CD3 epsilon, a T-cell specific surface antigen. Application of this BFA with human peripheral blood lymphocytes exhibited specific anti-tumor activity in vitro on a breast tumor cell line, ZR-75-1, which expressed abundant c-erbB-2 gene product on its cell surface. These results indicate that BFA recognizing an oncogene product on cell surface is a potential new agent for cancer immunotherapy.

The 20 kDa homologous restriction factor (HRF20, CD59) prevents formation of membrane attack complexes of human complement. Transfection of the cDNA of HRF20 to heterologous cells, which are sensitive to human complement, thereby renders these cells resistant to human complement attack. This property can then be used as a selection marker of transfection. In addition, the procedure is short and can be performed within 8 h. Since only 1 h is required for incubation with complement, the damage to the transfected cells is limited compared with damage resulting from selection for drug resistance which requires 10 days or more of incubation in the presence of harmful agents.

Vaccinia virus has been shown to render mouse tumor cells highly immunogenic. Since we have demonstrated that induction of complement activating capacity on guinea pig tumor cells by Sendai virus infection causes the tumor cells to become immunogenic, we assumed that vaccinia virus infection of mouse tumor cells might render them reactive with homologous mouse complement. Therefore, murine tumor cells, MH134 and X5563, infected with vaccinia virus (VV) were incubated with mouse plasma and C3 deposition was determined by staining with fluorescein isothiocyanate‐labeled anti‐C3. We found that VV‐infected tumor cells possess the ability to activate the alternative complement pathway (ACP) of murine complement. For induction of complement activating ability, at least a 3 h incubation of the infected MH134 cells was required indicating that the generation of ACP‐activating capacity on MH134 infected with VV is time‐dependent. Furthermore, ultraviolet‐irradiated vaccinia virus was able to induce ACP‐activating capacity on tumor cells as well.

Non-specific activation of complement (NAC) on cell membranes via the alternative pathway was studied by using leukemic cells and cells from a generalized reticulohistiocytosis. The cells were treated with normal human serum in veronal-buffered saline containing ethyleneglycoltetraacetrate and MG++. Since human erythrocytes (HuE) are known to adhere to complement-reacted cell membranes in an immune adherence reaction, complement activation on the cell membrane was confirmed by the rosette formation of HuE which is due to the generation of C3b molecules on the cell membrane. Only cells from Schilling-type acute monocytic leukemias and cells from a generalized reticulohistiocytosis possessed NAC ability. All other leukemic cells tested, as well as normal hematopoietic and lymphoreticular cells, were NAC-negative. Furthermore, none of the mitogens tested generated NAC ability on normal peripheral blood lymphocytes.