A human monoclonal antibody, produced following in vitro immunization, recognizing an epitope shared by HLA-A2 subtypes and HLA-A28.

In vitro immunization and subsequent immortalization of peripheral blood cells of a multiparous woman has resulted in the production of a stable human mouse heterohybridoma, 5C2A2, secreting an HLA-A2/A28-specific human monoclonal antibody. Although possibly exposed to HLA-A2 by transfusions, the cell donor showed no HLA-A2-specific serum antibodies. The present protocol for in vitro immunization includes the elimination of suppressor cells from the responder cell population, the presence of irradiated allogeneic lymphocytes as a source of antigen, as well as stimuli--recombinant interleukin-2 and a B-cell specific nucleoside analogue--causing the proliferation of B lymphocytes, prior to immortalization. The ability of the antibody 5C2A2 to detect all known HLA-A2 subtypes, except A2.3, and A28, allows identification of the serological epitope on the HLA-A2 molecule. Application of this in vitro immunization method allows the production of a set of HLA monoclonal antibody-secreting human hybridomas, independent of the existence of serum HLA antibodies in the lymphocyte donors.

Characterization of two human monoclonal antibodies reactive with HLA-B12 and HLA-B60, respectively, raised by in vitro secondary immunization of peripheral blood lymphocytes.

We have developed an in vitro immunization system for the production of B-cell lines that secrete HLA-specific human mAbs. For this purpose, peripheral blood lymphocytes of parous women were stimulated with pools of allogeneic lymphocytes. Preferential outgrowth of B-lymphocytes was effected by inclusion of rIL-2 and a B-cell specific nucleoside analogue. Stimulated B cells were immortalized by EBV transformation, and specific antibody-producing transformants were fused to heteromyeloma or mouse myeloma cell lines, yielding stable hybridomas. This approach has led to the successful development of two human heterohybridomas producing HLA-specific mAbs reactive by complement-mediated cytotoxicity. The specificities of these human mAbs, reactive with HLA-B12(44 + 45) and HLA-B60, respectively, are fully concordant with those of HLA-typing sera.

Successful immunotherapy of murine melanoma metastases with 7-thia-8-oxoguanosine.

We have recently reported that a synthetic nucleoside, 7-thia-8-oxoguanosine (7T8OG) is a potent activator of a number of effectors which are involved in anti-tumor immune responses. 7T8OG was found to induce interferon (IFN) production, to activate asialo-GM1 positive (AGM+1) killer cells, and to enhance specific antibody responses. In the present study, we investigated the effect of 7T8OG on growth of the murine pulmonary B16 melanoma and on formation of metastases. C57BL/6 mice were injected i.p. with 50-150 mg/kg 7T8OG before or after i.v. inoculation of B16 melanoma tumor cells, and 17-19 days after tumor inoculation, the number of metastases in the lungs were counted. 7T8OG given systemically in a single or a divided dose 24 h prior to the challenge of tumor cells reduced the number of lung tumor metastases by 89-99% which is highly significant as compared to untreated control (P less than 0.001). Occasional extra pulmonary tumor growth in the thoracic cavity and neck lymph node was also completely inhibited. The reduction in the number of tumor nodules was dose dependent. A single dose of 150 mg/kg of 7T8OG was also effective in inhibiting the growth of 3-5 day old metastatic tumors. The cytotoxic activity of killer cells induced in vivo by 7T8OG was completely abolished by in vitro treatment of cells with anti-AGM1 antibody plus complement. Administration of anti-AGM1 antibody following the 7T8OG treatment completely abrogated the anti-tumor effect of 7T8OG, resulting in a massive increase in the number of tumor foci in the lungs. Administration of carageenan or silica followed by injection of 7T8OG caused a significant increase (P less than 0.01) in the number of pulmonary tumor nodules compared to treatment with 7T8OG only. These findings indicate that activated macrophages or perhaps their cytokine (tumor necrosis factor) also contribute to the host tumor defense by 7T8OG.

Immunoenhancing properties and antiviral activity of 7-deazaguanosine in mice.

The nucleotide analog 7-deazaguanosine has not previously been reported to possess biological (antiviral or antitumor) properties in cell culture or in vivo. Up to 10(5) U of interferon per ml was detected in mouse sera 1 to 4 h following oral (200-mg/kg of body weight) and intraperitoneal (50-mg/kg) doses of the compound. 7-Deazaguanosine also caused significant activation of natural killer and phagocytic cells but did not augment T- and B-cell blastogenesis. Intraperitoneal treatments of 50, 100, and 200 mg/kg/day administered 24 and 18 h before virus inoculation were highly protective in mice inoculated with lethal doses of Semliki Forest or San Angelo viruses. Less but still significant survivor increases were evident in treated mice infected with banzi or encephalomyocarditis viruses. In most cases, the degree of antiviral activity was similar to that exhibited by the biological response modifier 7-thia-8-oxoguanosine. 7-Thia-8-oxoguanosine was more potent than 7-deazaguanosine against encephalomyocarditis virus in mice, however. Oral efficacy was achieved with 7-deazaguanosine treatments of greater than or equal to 100 mg/kg against all virus infections, whereas 7-thia-8-oxoguanosine is reported to be devoid of oral activity in rodents. Thus, 7-deazaguanosine represents the first reported orally active nucleoside biological response modifier exhibiting broad-spectrum antiviral activity against particular types of RNA viruses.

Potentiation of the efficacy of murine L1210 leukemia vaccine by a novel immunostimulator 7-thia-8-oxoguanosine: increased survival after immunization with vaccine plus 7-thia-8-oxoguanosine.

We have investigated the ability of a novel immunopotentiator, 7-thia-8-oxoguanosine (7T8OG) to increase the efficacy of a weakly immunogenic murine L1210 leukemia vaccine. The vaccine was prepared by irradiating L1210 leukemia cells in a cesium source with a total of 6000-R dose. DBA/2 mice were treated with 150 mg/kg 7T8OG and/or with vaccine consisting of 10(7) irradiated cells. In combination therapy, mice first received the vaccine and then were injected with 75 mg/kg 7T8OG 2 h and 4 h after vaccination. One week after the last treatment all mice were inoculated with 10(4) live leukemia cells intraperitoneally. Control, untreated mice (n = 66) injected with 10(4) live leukemia cells had a mean survival time +/- standard error of 10.5 +/- 0.2 days. Treating mice (n = 66) with one, two or three doses of 7T8OG administered i.p. 1 week apart did not increase survival (mean survival time = 10.7 days). Mice immunized with one, two or three doses of vaccine had 14.5 +/- 1.1, 45.4 +/- 6.2 and 68.3 +/- 10.6 days mean survival, respectively. 7T8OG-stimulated vaccination increased the survival dramatically. The best survival was noted when the mice were treated with 2x (vaccine + 7T8OG). Immunization of mice (n = 30) with this treatment regimen increased the mean survival to 156 +/- 10.0 days. Over 90% of mice that were treated this way had a cumulative survival time greater than 160 days. In contrast, only 12% of the mice immunized twice with the leukemia vaccine alone survived over 160 days. These results suggest a rationale for the use of this immuno-potentiator with various vaccines for a more effective immunization.

Guanosine analogues. Synthesis of nucleosides of certain 3-substituted 6-aminopyrazolo[3,4-d]pyrimidin-4(5H)-ones as potential immunotherapeutic agents.

Several guanosine analogues were synthesized in the pyrazolo[3,4-d]pyrimidine ring system with various substituents at the 3-position. The new analogues prepared here include the CH3 (2-amino-3-methyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13a), the phenyl (2-amino-3-phenyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13b), and the NH2 (3,6-diamino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4(5H)- one, 17) substituted derivatives. These new agents, as well as several other 3-substituted derivatives including H, Br, OCH3, COOH, and oxo, were evaluated for their ability to potentiate certain murine immune functions relative to the known active agent 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (4, 7-thia-8-oxoguanosine). The biological evaluation included the (1) ex vivo determination of increased natural killer cell function and (2) in vivo antiviral protection against a lethal challenge of Semliki Forest virus. The 3-unsubstituted (5a) and the 3-bromo (5c) derivatives were found to be the most active immunopotentiators in this series.

A novel guanosine analog, 7-thia-8-oxoguanosine, enhances macrophage and lymphocyte antibody-dependent cell-mediated cytotoxicity.

Intraperitoneal treatment of mice with a novel guanosine analog, 7-thia-8-oxoguanosine (7-thia-8-oxoGuo), gives rise to activated splenic lymphocytes and peritoneal macrophages with enhanced capacity to mediate antibody-dependent cellular cytotoxicity (ADCC). ADCC activities against both chicken red blood cells and P815 murine plasmacytoma cells were enhanced, indicating that macrophages as well as lymphocytes functioning as K-cells in the two distinct cytolytic systems, were activated by 7-thia-8-oxoGuo. Furthermore, 7-thia-8-oxoGuo enhanced lymphocyte-mediated ADCC activity in beige (bgJ/bgJ) mice against P815, thus indicating the ability of 7-thia-8-oxoGuo to function as a potent immunomodulator even in an animal that is known to possess selective impairment of naturally occurring killer lymphocytes. These results suggest that 7-thia-8-oxoGuo could serve as an agent for immunomodulation and immunorestoration.

Antiviral activity of the novel immune modulator 7-thia-8-oxoguanosine.

A novel thiazolopyrimidine nucleoside, 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (7-thia-8-oxoguanosine), was evaluated for antiviral activity in rodent models, and at 50-200 mg/kg prevented death in mice inoculated intraperitoneally (i.p.) with Semliki Forest, San Angelo, and banzi viruses when administered i.p. before virus challenge. Similarly, the nucleoside was effective against an intranasal challenge of rat coronavirus in suckling rats, with activity present when treatment started as late as 4 h after virus inoculation. Protection was observed against herpes type 1 and murine cytomegalovirus (both inoculated i.p.) infections, and encephalitis induced by intracerebral inoculation of a human coronavirus in mice. Friend leukemia virus splenomegaly was more severe in drug-treated animals than in placebos. This immune modulator is promising for the treatment of animal and human diseases.

Roles of interferon and natural killer cells in the antiviral activity of 7-thia-8-oxoguanosine against Semliki Forest virus infections in mice.

7-Thia-8-oxoguanosine is a novel biological response modifier with broad-spectrum antiviral activity against many DNA and RNA viruses in vivo. Since two of its properties are to induce interferon and to activate natural killer (NK) cells, we investigated the roles of the lymphokine and NK cells in the antiviral activity of the compound against Semliki Forest virus. Antibody to interferon alpha/beta could completely abolish the protective activity of the nucleoside against virus infection in mice, whereas antibodies to interferons beta and gamma could not, indicating that interferon alpha was of major importance to confer protection to the animals. Reduced activation of NK cells was also observed in mice treated with 7-thia-8-oxoguanosine and antibody to interferon alpha/beta. The role of NK cells in the protective activity of the compound was directly assessed in beige mice or in Swiss Webster mice treated with asialo GM1 antibody. In both experiments, the animals were protected from lethal virus infection by treatment with nucleoside. Spleen cells primed by 7-thia-8-oxoguanosine and adoptively transferred to untreated mice could not save them from virus-induced mortality. These three results provide evidence that natural killer cells activated by 7-thia-8-oxoguanosine play a minimal role in protection from acute Semliki Forest virus infections in mice.

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells.

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.

Growth inhibition and induction of cellular differentiation of human myeloid leukemia cells in culture by carbamoyl congeners of ribavirin.

A series of 1,2,3-triazole (2), pyrazole (3 and 5), and pyrrole (4) ribonucleosides with two adjacent carbamoyl groups have been synthesized and evaluated for cell growth inhibition and induction of cellular differentiation of HL-60 cells in culture. Glycosylation of the TMS derivatives of dimethyl 1,2,3-triazole-4,5-dicarboxylate (6) and diethyl pyrazole-3,4-dicarboxylate (7) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D- ribofuranose (8) in the presence of TMS triflate gave predominantly the beta-nucleosides 9 and 14, respectively. Ammonolysis of 9 and 14 furnished 2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide (2) and 1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide (3), respectively. Stereoselective ring annulation of 1-deoxy-1-hydrazinyl-2,3-O-isopropylidene-D- ribose (16) with tetracyanoethylene (15) gave 5-amino-1-(2,3-O-isopropylidene-beta-D-ribofuranosyl)pyrazole-3,4- dicarbonitrile (17). Deisopropylidenation of 17, followed by oxidative hydrolysis of the reaction product (18), gave the 5-amino derivative of 3 (5). Stereospecific glycosylation of the sodium salt of preformed diethyl pyrrole-3,4-dicarboxylate (22) with 1-chloro-2,3-O-isopropylidene-5-O-(tert-butyldimethylsilyl)-alpha-D- ribofuranose (23) was accomplished to furnish blocked nucleoside 24, which on ammonolysis and deisopropylidenation gave 1-beta-D-ribofuranosylpyrrole-3,4-dicarboxamide (4). The structures of 2 and 3 were assigned by single-crystal X-ray diffraction studies, which showed extensive inter- and intramolecular hydrogen bonding. Nucleosides 2-5 are devoid of significant cytotoxic properties against L1210 and WI-L2 leukemia cells in culture. However, these compounds were found to be inducers of cellular differentiation of HL-60 cells in the range of 30-60 microM and were comparable to ribavirin in this regard.

Thiazolo[4,5-d]pyrimidine nucleosides. The synthesis of certain 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidines as potential immunotherapeutic agents.

Novel analogues of the naturally occurring purine nucleosides were synthesized in the thiazolo[4,5-d]pyrimidine ring system to determine the immunomodulatory effects of insertion of a sulfur atom in place of nitrogen at position 7 of the purine ring. In particular, 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (7, guanosine analogue), 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,5,7(3H,4H,6H) trione (8, xanthosine analogue), 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione (10, inosine analogue), and 7-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidin-2(3H)-one (32, adenosine analogue) were prepared, as well as the 8-mercaptoguanosine (14) and 6-mercaptoguanosine (17) analogues. Single-crystal X-ray studies confirmed the structural assignment of 17 and 32 as having the beta-configuration with the site of glycosylation at N3. The nucleosides were evaluated for their ability to potentiate various murine immune functions in direct comparison to the known active agents 8-bromoguanosine (1), 8-mercaptoguanosine (2), and 7-methyl-8-oxoguanosine (3). Two of the guanosine analogues, 7 and 14, were found to exhibit significant immunoactivity relative to the positive control compounds (1-3), while the adenosine, inosine, xanthosine, and 6-mercaptoguanosine analogues were devoid of activity. Compound 7 exhibited greater immunoactivity than any of the other guanosine analogues and derivatives in all test systems. Specifically, 7 was shown to be about twice as potent as 3 in the murine spleen cell mitogenicity assay. In addition, treatment with 7 produced about a 4-fold increase in natural killer cell cytotoxicity, while treatment with 3 afforded a 3-fold increase over controls. Finally, 7 provided excellent protection (92% survivors compared to 0% for placebo controls) against Semliki Forest virus in mice. Induction of interferon may account for the major mode of action of these guanosine analogues.

Baboon-to-human cardiac xenotransplantation in a neonate.

This report details the first case of cardiac xenotransplantation in a neonate. The recipient, a victim of hypoplastic left heart syndrome (HLHS), survived 20 days. Autopsy findings are documented. The cardiac graft showed only traces of cell-mediated rejection. Graft failure appears to have resulted from a progressive, potentially avoidable humoral response, unmodified by immunosuppression. Cardiac allotransplantation and selective baboon-to-human xenotransplantation deserve further exploration as investigational therapy for neonatal HLHS.

KIE: The first transplantation of a baboon heart into a newborn (Baby Fae) with hypoplastic left heart syndrome (HLHS) is reported by the team involved in the operation. Details of the pretransplant immunologic testing of the organ recipient and potential baboon donors, and the pre-and post-operative course of the patient until her death 20 days after surgery are discussed. Autopsy findings showed only traces of cell-mediated rejection. Graft failure appears to have resulted from a progressive, potentially avoidable humoral response, unmodified by immunosuppression, perhaps as a result of crossing the ABO blood barrier. The authors believe that cardiac replacement by xenotransplantation is technically feasible and a reasonable investigative option for newborns with HLHS.

Orthotopic cardiac xenografting in the newborn goat.

Fourteen newborn (less than 7 days) goats were subjected to orthotopic cardiac transplantation with donor xenografts from size-matched lambs. Ten goats survived the operation (greater than 24 hours). Recipient animals received cyclosporine 48 and 24 hours before the operation and daily after the operation on a gradually reducing daily protocol. Recipients were also given pulse doses of methylprednisolone (100 mg/kg) and azathioprine (3 mg/kg) once a week, the dosage schedule being gradually reduced and azathioprine discontinued as recipients became long-term survivors (greater than 60 days). Seven recipients had radionuclide left ventriculography for measurement of left ventricular ejection fraction from 1 to 4 months postoperatively. Ejection fractions at 1 month in two recipients were 35% and 57%, and at 2 months the ejection fraction in one recipient was 58%. Serial ejection fractions from 1 to 4 months postoperatively in four recipients averaged 50%, 58%, 45%, and 45%. Survival in days among the 10 recipients was 24, 32, 44, 47, 60, 60, 78, 90, 120, and 165. Average survival was 72 days. There were no significant infections. Most animals showed mild-to-moderate subacute and chronic graft rejection at autopsy. One host showed no gross or microscopic graft rejection at autopsy on postoperative day 47. Tumor was not observed. These data suggest that long-term survival may be feasible for newborn recipients of cardiac xenografts with cyclosporine therapy and limited supplemental immunosuppression.

Islet autotransplantation after pancreatectomy for chronic pancreatitis with a new method of islet preparation.

Near-total (95 percent) pancreatectomy with intraportal islet autotransplantation was performed in five patients with chronic pancreatitis. Two patients are completely insulin-independent 7 and 14 months after autotransplantation; the other three patients obtained partial independence. A new method of islet cell preparation is described. Islet cell autotransplantation after total or near-total pancreatectomy should at present be viewed with cautious optimism. Additional cases need to be studied and followed up before its role in the surgery of chronic pancreatitis will be clear.