Postexposure prevention of progressive vaccinia in SCID mice treated with vaccinia immune globulin.

A recently reported case of progressive vaccinia (PV) in an immunocompromised patient has refocused attention on this condition. Uniformly fatal prior to the licensure of vaccinia immune globulin (VIG) in 1978, PV was still fatal in about half of VIG-treated patients overall, with a greater mortality rate in infants and children. Additional therapies would be needed in the setting of a smallpox bioterror event, since mass vaccination following any variola virus release would inevitably result in exposure of immunocompromised people through vaccination or contact with vaccinees. Well-characterized animal models of disease can support the licensure of new products when human studies are not ethical or feasible, as in the case of PV. We chose vaccinia virus-scarified SCID mice to model PV. As in immunocompromised humans, vaccinia virus-scarified SCID animals develop enlarging primary lesions with minimal or no inflammation, eventual distal virus spread, and lethal outcomes if left untreated. Postexposure treatment with VIG slowed disease progression, caused local lesion regression, and resulted in the healthy survival of most of the mice for more than 120 days. Combination treatment with VIG and topical cidofovir also resulted in long-term disease-free survival of most of the animals, even when initiated 7 days postinfection. These results support the possibility that combination treatments may be effective in humans and support using this SCID model of PV to test new antibody therapies and combination therapies and to provide further insights into the pathogenesis and treatment of PV.

Establishing efficacy of human products using animals: the US food and drug administration's "animal rule".

In 2002, the US Food and Drug Administration issued regulations to allow the approval of human drugs and biological products based on animal efficacy studies when human efficacy studies would be unethical or not feasible. These regulations are intended to assist in the approval process for products aimed at preventing or treating human diseases caused by nuclear, radiological, biological, and chemical agents that have the potential to harm a significant percentage of the US population. This article discusses the criteria that must be met to use the Animal Rule to demonstrate efficacy in place of human clinical trials.

Biobehavioural correlates of hand preference in free-ranging female primates.

In this research we examined biological and behavioural correlates of handedness in a subject cohort of 41 free-ranging young female rhesus macaques (Macaca mulatta). Specifically, we examined relationships between handedness and cerebrospinal fluid (CSF) concentrations of the monoamine metabolites 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), plasma concentrations of the hormones cortisol and adrenocorticotropin (ACTH), prolactin, and multiple indices of social behaviour, including proximity to other animals, grooming, submission, and aggression. Handedness was determined through systematic observation of animals reaching for food in their unrestricted home environment. We found a population-level bias for left-hand use in this cohort of young females. The frequency of right versus left hand use was positively correlated with CSF 5-HIAA, plasma cortisol concentrations, the frequency of submissive behaviour, and with the frequency of bouts in which animals received low-level aggression. The positive correlation between right versus left hand use, submissive behaviour, and received aggression found here in females contrasts with the negative correlation among these same variables that we have previously reported in rhesus males. We conclude that these results may be explicable in terms of sex-based differences in rhesus life-history patterns, and that the influence of the serotonergic system on patterns of male aggression, social behaviour, and handedness, and the associations between handedness and social behaviour found previously among males may not be generalised to female rhesus macaques.

In vivo overexpression of IL-13 receptor alpha2 chain inhibits tumorigenicity of human breast and pancreatic tumors in immunodeficient mice.

Interleukin 13 receptor alpha2 (IL-13R(alpha)2) chain is highly expressed on some tumor cell lines and primary cell cultures. This receptor chain plays an important role in ligand binding and internalization. To determine the functional significance of overexpression of this chain, we stably transfected IL-13R(alpha)2 chain in human breast (MDA-MB-231) and pancreatic (PANC-1) cancer cell lines that naturally do not express this chain. There was no difference in growth between vector only transfected and IL-13R(alpha)2 chain transfected cells in vitro. However, surprisingly, in immunodeficient mice, tumorigenicity was profoundly inhibited in IL-13R(alpha)2 chain overexpressing tumors. Because breast tumors that grew later showed loss of IL-13R(alpha)2 gene expression, lack of tumorigenicity correlated positively with IL-13R(alpha)2 chain expression. Inflammatory cells including neutrophils and macrophages were identified in IL-13R(alpha)2 overexpressing regressing tumors and neutrophils were found to produce IL-13. IL-13 showed a modest antitumor activity to IL-13R(alpha)2 chain overexpressing tumors in vitro and in vivo. Furthermore, IL-13R(alpha)2 chain overexpressing tumors constitutively produced IL-8 that has been shown to have antitumor effect. These results establish a novel function of a cytokine receptor chain and further suggest that the presence of this chain on tumor cells by itself may play a key role in tumorigenicity.

Evaluation of a neonatal rat model for prediction of mumps virus neurovirulence in humans.

Neurovirulence of several mumps virus strains was assessed in a prototype rat neurovirulence test and compared to results obtained in the monkey neurovirulence test. The relative human neurovirulence of these strains was proportional to the severity of hydrocephalus in rats but not to lesion scores in the monkeys.

The mumps virus neurovirulence safety test in Rhesus monkeys: a comparison of mumps virus strains.

Wild type mumps viruses are highly neurotropic and a frequent cause of aseptic meningitis in unvaccinated humans. To test whether attenuated mumps viruses used in the manufacture of mumps vaccines have neurovirulent properties, a monkey neurovirulence safety test (MNVT) is performed. However, results with several mumps virus MNVTs have raised questions as to whether the test can reliably discriminate neurovirulent from nonneurovirulent mumps virus strains. Here, various mumps virus strains representing a wide range of neuropathogenicity were tested in a standardized MNVT. A trend of higher neurovirulence scores was observed in monkeys inoculated with wild type mumps virus versus vaccine strains, although differences were not statistically significant. Results indicated the need for further examination and refinement of the MNVT or for development of alternative MNVTs.

Preclinical evaluation of group B Neisseria meningitidis and Escherichia coli K92 capsular polysaccharide-protein conjugate vaccines in juvenile rhesus monkeys.

We reported the first use of group B meningococcal conjugate vaccines in a nonhuman primate model (S. J. N. Devi, C. E. Frasch, W. Zollinger, and P. J. Snoy, p. 427-429, in J. S. Evans, S. E. Yost, M. C. J. Maiden, and I. M. Feavers, ed., Proceedings of the Ninth International Pathogenic Neisseria Conference, 1994). Three different group B Neisseria meningitidis capsular polysaccharide (B PS)-protein conjugate vaccines and an Escherichia coli K92 capsular polysaccharide-tetanus toxoid (K92-TT) conjugate vaccine are here evaluated for safety and relative immunogenicities in juvenile rhesus monkeys with or without adjuvants. Monkeys were immunized intramuscularly with either B PS-cross-reactive material 197 conjugate, B PS-outer membrane vesicle (B-OMV) conjugate, or N-propionylated B PS-outer membrane protein 3 (N-pr. B-OMP3) conjugate vaccine with or without adjuvants at weeks 0, 6, and 14. A control group of monkeys received one injection of the purified B PS alone, and another group received three injections of B PS noncovalently complexed with OMV. Antibody responses as measured by enzyme-linked immunosorbent assay varied among individual monkeys. All vaccines except B PS and the K92-TT conjugate elicited a twofold or greater increase in total B PS antibodies after one immunization. All vaccines, including the K92-TT conjugate, elicited a rise in geometric mean B PS antibody levels of ninefold or more over the preimmune levels following the third immunization. Antibodies elicited by N-pr. B-OMP3 and B-OMV conjugates were directed to the N-propionylated or to the spacer-containing B PS antigens as well as to the native B PS complexed with methylated human serum albumin. None of the vaccines caused discernible safety-related symptoms.

Preclinical development of a recombinant toxin containing circularly permuted interleukin 4 and truncated Pseudomonas exotoxin for therapy of malignant astrocytoma.

Effective treatment is lacking for malignant glioblastoma/astrocytoma. We have identified interleukin-4 receptors (IL-4R) on human malignant astrocytoma. We demonstrate that 16 of 21 surgical samples of high-grade astrocytoma and glioblastoma but not normal brain tissues expressed IL-4R as assessed by reverse transcriptase PCR. We further demonstrate that human malignant astrocytoma cell lines express high-affinity IL-4R. Using a chimeric protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin A, we observed that this toxin IL4(38-37)-PE38KDEL) is highly cytotoxic to IL-4R-bearing glioblastoma cells. Compared with a previously reported IL4-PE chimeric protein (IL-PE4E), IL4(38-37)-PE38KDEL bound with higher affinity and was 3-30-fold more cytotoxic to glioblastoma cell lines. Upon intrathecal administration in monkeys, high cerebrospinal fluid IL4(38-37)-PE38KDEL levels were achieved using 2- and 6-microg/kg doses without any central nervous system or other abnormalities. IL4(38-37)-PE38KDEL levels were not detectable in the serum of any monkey studied. When IL4(38-37)-PE38KDEL was injected into the right frontal cortex of rats, localized necrosis was observed at 1000-ng/ml doses but not at < or = 100-ng/ml doses. We conclude that by localized administration, nontoxic levels of IL4(38-37)-PE38KDEL can be achieved, which may have significant cytotoxic activity against malignant astrocytoma.

Biological properties of chimeric domain-deleted anticarcinoma immunoglobulins.

CC49 is a second-generation monoclonal antibody (MAb) that has high affinity for the tumor-associated pancarcinoma antigen tumor-associated glycoprotein-72. In clinical trials using gamma scanning, radiolabeled CC49 has facilitated the detection of more than 90% of carcinomas. We report here the development of a constant heavy-chain 2 (CH2) domain-deleted chimeric (c) CC49 MAb by transfecting an expression construct consisting of the CC49 murine variable region and a CH2 domain-deleted human IgG1 constant region into cCC49 kappa producing SP2/0 murine myeloma cells. As determined by SDS-PAGE, the intact cCC49 delta CH2 has a molecular weight of 153,000 and, under reducing conditions, molecular weights of 43,000 and 27,000. The plasma clearance and tumor-targeting properties of cCC49 delta CH2 were evaluated and compared with those of mouse/human chimeric forms cCC49 delta CH1 and intact cCC49. Previous studies have shown that the in vitro antigen-binding properties of cCC49 delta CH1 are similar to those of cCC49. Biodistribution studies reported here, using 131I-labeled cCC49 delta CH1 and 125I-labeled cCC49 in athymic mice bearing human colon carcinoma xenografts, demonstrated that both cMAbs localized to the tumor and cleared from the normal tissues similarly. However, in comparison with 125I-labeled cCC49, 131I-labeled cCC49 delta CH2 localized to tumors earlier and had a significantly lower percentage of the injected dose of cMAb/g (%ID/g) in normal tissues than cCC49. Immunoscintigraphy of 131I-labeled cCC49 delta CH2 and 125I-labeled cCC49 in athymic mice bearing human tumor xenografts demonstrated a clear image of the tumor by 24 h after i.v. administration of the delta CH2 cMAb versus the 72 h required for cCC49. Biodistribution studies using 177Lu-conjugated cCC49 delta CH1 and cCC49 showed no significant difference between the radiolocalization indices (% ID/g in tumor divided by % ID/g in normal tissue). 177Lu-conjugated cCC49 delta CH2, however, had lower % ID/g values in tumor xenografts and lower radiolocalization indices than either 177Lu-conjugated cCC49 delta CH1 or 177Lu-conjugated cCC49. Pharmacokinetic studies in non-tumor-bearing athymic mice using cCC49 delta CH1 and cCC49 revealed no significant difference between these cMAbs. However, the plasma clearance of cCC49 delta CH2 in non-tumor-bearing mice was significantly faster than that of cCC49. These results were similar when the cMAbs were labeled with either iodine or lutetium. In nonhuman primates, 131I-labeled cCC49 delta CH2 cleared significantly faster than 125I-labeled cCC49. The similar plasma clearance and tumor localization of cCC49 and cCC49 delta CH1 suggest that these two cMAbs may be used in similar clinical settings. However, because of the unique pharmacokinetics and tumor targeting of cCC49 delta CH2 versus cCC49 or cCC49 delta CH1, this chimeric immunoglobulin form may be useful in clinical settings that require efficient tumor targeting and rapid serum and whole-body clearance.

Experimentally induced septic arthritis in chimpanzees infected with Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma urealyticum.

Mycoplasma hominis was isolated in pure culture from septic synovial aspirates from an individual (patient A) during 16 different bouts of exacerbation over a 70-month period of observation. Two isolates, 10(7) and also 10(6) color-changing units (CCU) of the 1620 isolate and 5 x 10(4) CCU of the 1628 isolate, caused inflammation in chimpanzees inoculated intraarticularly. Inflammation was also induced with 10(7) CCU of the 2010B isolate, serovar VII of Ureaplasma urealyticum, recovered from an agammaglobulinemic individual (patient B) with septic polyarthritis and with 3 x 10(6) CCU of the PI-1428 isolate of Mycoplasma pneumoniae. Inflammation persisted for up to 36 days and was self-limiting. The aspirates contained up to 220,000 white blood cells/mm3 and up to 10(7) CCU/mL. There was good correlation between the severity of inflammation and the numbers of organisms, but antibody was not detected in aspirates during the peak severity of disease. As the numbers of organisms, decreased, detectable levels of antibody increased, thus suggesting that antibody may have been bound to antigen. Chimpanzees previously infected with either the 1628 isolate of M. hominis or the 2010B isolate of U. urealyticum were protected on challenge with > 100 times the minimal dose causing arthritis. Chimpanzees showed little or no inflammation when inoculated intraarticularly with 5 x 10(8) CCU of the type strain PG-21 of M. hominis or with the type strain CO of U. urealyticum or when inoculated intravenously with 3 x 10(8) CCU of the arthrogenic 1620 isolate of M. hominis.

The effect on the safety of intravenous immunoglobulin of testing plasma for antibody to hepatitis C.

BACKGROUND: The safety of intravenous immunoglobulin (IGIV), manufactured from units testing negative for antibody to hepatitis C virus (anti-HCV), was investigated. STUDY DESIGN AND METHODS: A study involving five chimpanzees was performed to determine whether the safety of IGIV would be compromised if units of plasma that reacted for anti-HCV were withheld from pools from which IGIV is manufactured. In the first phase of the experiment, two chimpanzees were infused with 25 mL per kg of unprocessed, pooled plasma from 2887 donors who did not react for anti-HCV in single-antigen (c100-3) enzyme-linked immunosorbent assays. In the second phase, each of three chimpanzees was infused with 1000 mg per kg of IGIV manufactured from the same plasma units. The immunoglobulin was made by seven United States-licensed manufacturers, each using its own approved method. Each chimpanzee received an equal dose of each manufacturer's IGIV. RESULTS: The two chimpanzees that received anti-c100-3-nonreactive, unprocessed pooled plasma became infected with HCV. The three chimpanzees infused with IGIV did not show any evidence of infection with HCV 15 months after inoculation. Two of these animals were challenged with human non-A,non-B hepatitis-infectious plasma, and both subsequently showed evidence of HCV infection. CONCLUSION: These studies demonstrate that, as determined by infectivity for chimpanzees, 1) the withholding of plasma units that react for anti-c100-3 from pools from which plasma products are manufactured does not render the source material noninfectious, and 2) the safety of IGIV manufactured from such plasma pools is not compromised by withholding the units that react for anti-c100-3.

Pertussis toxin-induced alterations of murine hepatic drug metabolism following administration of diphtheria and tetanus toxoids and pertussis vaccine adsorbed.

Administration of pertussis toxin (PT) in combination with diphtheria and tetanus toxoids adsorbed (DT vaccine) or with acellular pertussis vaccine adsorbed and diphtheria and tetanus toxoids (APDT) elicits dose- and time-dependent alterations in hepatic drug metabolism in mice. Cytochrome P-450 (P-450) levels were inhibited more than 50% at 7 days following a single injection of PT mixed with either vaccine. When combined with DT vaccine, 125 ng of PT was required to produce this effect, while as little as 16 ng of PT combined with APDT vaccine inhibited P-450 levels. The inhibition of P-450 levels is similar to that observed after a single injection of diphtheria and tetanus toxoids and pertussis vaccine adsorbed (DTP). Alterations of P-450 levels were accompanied by increased activities of quinone reductase but not with changes in plasma interleukin-6 or tumor necrosis factor levels. Other Bordetella pertussis virulence factors, such as filamentous hemagglutinin, fimbriae and pertactin, were also tested but had no significant effect on hepatic drug metabolism. Endotoxin or preparations containing endotoxin caused alterations in hepatic drug metabolism within 24 h, concomitant with increased interleukin-6 and tumor necrosis factor levels, but these effects had resolved by 1 week. DTP vaccine and preparations containing PT caused a marked induction of gamma interferon coincident with the maximal inhibition of P-450 levels. This effect was not present with DT or APDT vaccine alone, nor with endotoxin or any combination of factors that did not contain PT. These results demonstrate that PT is a necessary component for the sustained effects of DTP vaccine on hepatic drug metabolism and suggest a role for gamma interferon in this process.

Immunogenicity and safety of a recombinant vaccinia virus vaccine expressing the carcinoembryonic antigen gene in a nonhuman primate.

We have previously reported the development of a recombinant vaccinia virus vaccine expressing the human carcinoembryonic antigen (CEA) gene, designated rV(NYC)-CEA. This construct has been shown to elicit specific anti-CEA immune responses and an antitumor effect in a murine tumor model. In the studies reported here, the safety and immunogenicity of this recombinant vaccinia virus were evaluated in a rhesus monkey model. Human CEA is a M(r) 180,000 glycoprotein expressed in approximately 90% of gastrointestinal carcinomas and in some breast and non-small cell lung carcinomas. This family also includes normal cross-reacting antigen (NCA). Rhesus monkeys, like humans, have some NCA on the surface of their granulocytes. Eight monkeys were immunized 3 or 4 times by skin scarification with the recombinant CEA vaccine and four monkeys received wild-type vaccinia virus as control. After three vaccinations, all rV(NYC)-CEA-vaccinated animals exhibited a strong anti-CEA antibody response as measured by enzyme-linked immunosorbent assay. The functional ability of these antibodies to mediate lysis of a CEA-bearing tumor cell was demonstrated using human effector cells. This response could be enhanced by interleukin 2. Cellular immunity to CEA was measured by delayed-type hypersensitivity upon intradermal challenge with purified CEA. Only those animals receiving the recombinant vaccine displayed significant anti-CEA responses. Furthermore, peripheral blood mononuclear cells from immunized monkeys were found to proliferate in response to CEA stimulation. All vaccinated monkeys developed local skin irritation at the site of the vaccination, regional lymphadenopathy, and low-grade fevers after immunization. Following immunization with rV(NYC)-CEA, the response was consistent with the usual constitutional symptoms seen with human smallpox virus immunization. Blood counts, differentials, and hepatic and renal chemistries remained normal in all animals throughout the study and for up to 1 year following the primary vaccination. No evidence of immunological cross-reactivity to NCA was found by either a fall in the granulocyte count or analyses for anti-NCA antibodies. Thus, the rV(NYC)-CEA vaccine appears to be safe in rhesus monkeys. The administration of a CEA recombinant vaccine to rhesus monkeys induces both a humoral and a cell-mediated immune response directed against human CEA.

Role of endotoxin in alterations of hepatic drug metabolism by diphtheria and tetanus toxoids and pertussis vaccine adsorbed.

Administration of diphtheria and tetanus toxoids and pertussis vaccine adsorbed (DTP vaccine) or endotoxin (LPS) resulted in marked alterations in hepatic drug-metabolizing enzymes in endotoxin-responsive (R) and non-endotoxin-responsive (NR) mice. A single human dose (0.5 ml) of DTP vaccine increased hexobarbital-induced sleep times to 1.6- to 1.8-fold above those of controls in both strains of mice. This effect persisted for 7 days. In contrast, Bordetella pertussis LPS-treated mice showed an increase at 1 day (3.0-fold for R mice and 1.5-fold for NR mice), which returned to control levels by day 7. Furthermore, cytochrome P-450 levels were decreased 30 to 40% 24 h after DTP vaccine administration in both R and NR mice, while after LPS administration they were decreased 30% in R mice and less than 10% in NR mice. Both spleen and liver weights of R and NR mice were increased 7 to 14 days following DTP vaccine administration. However, LPS treatment had no apparent effect on liver weights, and spleen weights of R mice were elevated from days 3 to 7. Histopathologic tissue examination showed random, multifocal inflammation with hepatocyte necrosis after DTP vaccine administration to both R and NR mice and an absence of lesions in LPS-treated mice. Premixing LPS with polymyxin eliminated the increased sleep times, but premixing DTP vaccine with polymyxin did not affect the increased sleep times. Levels of tumor necrosis factor and interleukin-6 in plasma of R mice were markedly increased after DTP and LPS treatment, while NR mice had reduced increases. These results suggest that LPS contributes to the alterations in R and NR mice seen within the first 24 h of vaccine administration but that it is not likely to contribute to the effects observed at later time points.

Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K-12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen.

A live, oral Shigella vaccine, constructed by transfer of the 140-MDa invasiveness plasmid from Shigella flexneri 5 and the chromosomal genes encoding the group- and type-specific O antigen of S. flexneri 2a to Escherichia coli K-12, was tested in humans. Designated EcSf2a-1, this vaccine produced adverse reactions (fever, diarrhea, or dysentery) in 4 (31%) of 13 subjects who ingested a single dose of 1.0 x 10(9) CFU, while at better-tolerated doses (5.0 x 10(6) to 5.0 x 10(7) CFU), it provided no significant protection against challenge with S. flexneri 2a. A further-attenuated aroD mutant derivative, EcSf2a-2, was then tested. Rhesus monkeys that received EcSf2a-2 in three oral doses of ca. 1.5 x 10(11) CFU experienced no increase in gastrointestinal symptoms compared with a control group that received an E. coli K-12 placebo. Compared with controls, the vaccinated monkeys were protected against shigellosis after challenge with S. flexneri 2a (60% efficacy; P = 0.001). In humans, EcSf2a-2 was well tolerated at inocula ranging from 5.0 x 10(6) to 2.1 x 10(9) CFU. However, after a single dose of 2.5 x 10(9) CFU, 4 (17%) of 23 subjects experienced adverse reactions, including fever (3 subjects) and diarrhea (209 ml) (1 subject), and after a single dose of 1.8 x 10(10) CFU, 2 of 4 subjects developed dysentery. Recipients of three doses of 1.2 to 2.5 x 10(9) CFU had significant rises in serum antibody to lipopolysaccharide (61%) and invasiveness plasmid antigens (44%) and in gut-derived immunoglobulin A antibody-secreting cells specific for lipopolysaccharide (100%) and invasiveness plasmid antigens (60%). Despite its immunogenicity, the vaccine conferred only 36% protection against illness (fever, diarrhea, or dysentery) induced by experimental challenge (P = 0.17). These findings illustrate the use of an epithelial cell-invasive E. coli strain as a carrier for Shigella antigens. Future studies must explore dosing regimens that might optimize the protective effects of the vaccine while eliminating adverse clinical reactions.

Relationship of neurologic status in macaques infected with the simian immunodeficiency virus to cerebrospinal fluid quinolinic acid and kynurenic acid.

Increased concentrations of the excitotoxin quinolinic acid (QUIN) have been implicated in the neurologic deficits and brain atrophy that may accompany infection with the human immunodeficiency virus type-1. Key neuropathologic features of the AIDS encephalitis are replicated in some macaques following infection with the simian immunodeficiency virus (SIV). In the present studies, cerebrospinal fluid (CSF) QUIN concentrations increased within 2 weeks following infection of 11 rhesus macaques (Macaca mulatta) with a neurotropic sooty mangabey isolate of the simian immunodeficiency virus (SIVsm) and were sustained to greater than 2 standard deviations above uninfected control macaques. Highest CSF QUIN concentrations (up to 400-fold above pre-inoculation levels) were observed in 6 SIVsm-infected macaques with motor and behavioral abnormalities during life, brain atrophy on MRI scan and inflammatory lesions within the brain and meninges. Four of the 6 neurologic macaques deteriorated rapidly within 12 weeks after inoculation and had substantially larger increases in CSF QUIN levels than 2 other neurologic macaques and 5 macaques without neurologic signs which survived for longer than 37 weeks. Increases in serum QUIN and CSF kynurenic acid also occurred but generally to a lesser degree than the increases in CSF QUIN. In some animals, increases in serum L-kynurenine concentrations and reductions in CSF and serum L-tryptophan occurred and were consistent with activation of indoleamine-2, 3-dioxygenase, the first enzyme of the kynurenine pathway in extrahepatic tissues. CSF QUIN exceeded serum QUIN in 8.8% of samples from macaques with neurologic signs, supporting increased QUIN synthesis within the central nervous system. Production of [13C6]QUIN was demonstrated in one SIVsm-infected macaque and one uninfected control macaque following an intracisternal injection of [13C6]L-tryptophan and suggests that L-tryptophan is a substrate for QUIN synthesis within the nervous system or meninges, although the cellular localization of QUIN synthesis remain to be determined. We conclude that increases in kynurenine pathway metabolism occur in SIV-infected macaques and are most prominent in macaques with neurologic signs. Macaques infected with SIV offer a model to investigate the relationship between the metabolism of neuroactive kynurenines and neurologic disturbances associated with retroviral infection.

Variable infections with and serologic responses of rabbits to five different HIV-1 strains, including one neural tissue isolate.

The infectivity of different strains of HIV-1 in rabbits was investigated. The HIV-1RF and HIV-1MN inocula induced anti-envelope antibodies detectable by Western blot, and in the case of HIV-1RF, these antibodies were also detectable by ELISA. The peripheral blood lymphocytes (PBL) and lymph nodes from rabbits inoculated with HIV-1IIIB, HIV-1MN and HIV-1Z3, were positive for virus by culture and by polymerase chain reaction (PCR). HIV-1BRVA, originally isolated from a patient with AIDS dementia, infected the brain of the inoculated rabbit, as indicated by both virus culture and PCR. In this case PCR was positive using four different primer pairs. Throughout the study, rabbits showed no clinical signs of HIV-1 infection and no remarkable histopathology was observed in the tissues examined. The apparent differences in infectivity and tissue tropism of the five HIV-1 strains demonstrated here provide additional evidence that the rabbit may serve as a useful model for studying HIV-1 infection and pathogenesis.

Comparative biological properties of a recombinant chimeric anti-carcinoma mAb and a recombinant aglycosylated variant.

It has been demonstrated previously that the degree of glycosylation of a molecule may alter its pharmacokinetic properties and, in the case of an antibody, its metabolism and other biological properties. Transfectomas producing aglycosylated chimeric B72.3(gamma 1) pancarcinoma monoclonal antibody (mAb) were developed by introduction of the eukaryotic expression construct pECMgpB72.3 HuG1-agly, into SP2/0 murine myeloma cells producing the chimeric kappa chain of mAb B72.3. After cell cloning, one subclone with the highest binding to the TAG-72-positive human colon carcinoma was designated mAb aGcB72.3, and its biological and biochemical properties were compared with those of the chimeric B72.3(gamma 1), designated mAb cB72.3. Polyacrylamide gel electrophoresis showed that under non-reducing conditions, the molecular masses of the aGcB72.3 and cB72.3 mAbs were 162 kDa and 166 kDa respectively. The heavy chain of mAb aGcB72.3 had a slightly faster mobility than that of cB72.3, while the mobility of the light chains of the two chimeric mAbs was similar. No difference was observed in the isoelectric points of either chimeric mAb. Liquid competition radioimmunoassays demonstrated that the aGcB72.3 and cB72.3 mAbs have comparable binding properties to TAG-72. These studies demonstrate that aglycosylation of the chimeric IgG1 mAb B72.3 at the CH2 domain, as has been shown for other mAbs [Dorai H., Mueller B., Reisfeld R. A., Gillies S. D. (1991) Hybridoma 10:211; Morrison S. L., Oi V. T. (1989) Adv Immunol 44:65], eliminates antibody-dependent cell-mediated cytotoxicity activity, but does not substantially alter affinity or plasma clearance in mice. These studies also demonstrate for the first time (a) no difference in plasma clearance of an aglycosylated and a chimeric mAb in a primate after i.v. inoculation; (b) a difference (P less than or equal to 0.05) in mice in the more rapid peritoneal clearance of a chimeric mAb versus an aglycosylated chimeric mAb; (c) higher (0.05 less than or equal to P less than or equal to 0.1) tumor: liver ratios at 24, 72 and 168 h using 111In-labeled aglycosylated chimeric mAb versus chimeric mAb. Since the liver is the major site of metastatic spread for most carcinomas, slight differences in tumor to normal liver ratios may be important in diagnostic applications. These studies thus indicate that comparative analyses of a novel recombinant construct (i.e., aglycosylated) and its standard chimeric counterpart require documentation in more than one system and are necessary if one is ultimately to define optimal recombinant/chimeric constructs for diagnosis and therapy in humans.