Simultaneous detection of multiple cytokines and chemokines from nonhuman primates using luminex technology

Cytokines and chemokines are soluble mediators of the immune system that play a crucial role in intercellular signaling, and in the recruitment of cells to inflammation sites. Identification of these molecules in nonhuman primates (NHP) is crucial for the understanding of complex physiological and pathological mechanisms that occur in these species, and to demonstrate whether these mechanisms function similarly in humans. The Luminex100 system is a bench-top flow cytometer that allows the user to perform up to 100 tests simultaneously in a single tube. Recently, a significant number of commercial vendors have developed kits for the simultaneous detection of multiple cytokines and chemokines of human origin with the Luminex system. These kits were tested for their capacity to recognize chemokines and cytokines of nonhuman primate origin. ELISA and ELISPOT assays were also adapted to the Luminex format, and novel assays based on new combinations of antibodies were developed. PBMC were isolated from blood from chimpanzees, rhesus macaques, baboons, cynomolgus macaques, pig-tailed macaques, and African green monkeys; these cells were stimulated in vitro and culture supernatants were used for the determination of cytokines and chemokines. Crossreactivity tables were prepared based on the ability of the reagents to detect cytokines and chemokines in NHP samples with similar intensity to the ones observed in human samples. By mixing commercially available reagents and newly developed ones, a combination has been created that allows for the detection of 20 NHP chemokines and cytokines in a single sample, including G-CSF, GM-CSF, IFN-gamma, IL-1beta, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 (p40), IL-17, IL-18, MCP-1, MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, and TNF-beta. These reagents may become a very useful resource for scientists working with these NHP species, which are relevant pre-clinical models for human diseases and transplantation because they approximate humans in physiology and genetics more closely than any other animal.     

Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis)

Anthrax is considered a serious biowarfare and bioterrorism threat because of its high lethality, especially by the inhalation route. Rhesus macaques (Macaca mulatta) are the most commonly used nonhuman primate model of human inhalation anthrax exposure. The nonavailability of rhesus macaques necessitated development of an alternate model for vaccine testing and immunologic studies. This report describes the median lethal dose (LD(50)) and pathology of inhalation anthrax in cynomolgus macaques (Macaca fascicularis). Gross and microscopic tissue changes were reviewed in 14 cynomolgus monkeys that died or were killed after aerosol exposure of spores of Bacillus anthracis (Ames strain). The LD(50) and 95% confidence intervals were 61800 (34000 to 110000) colony-forming units. The most common gross lesions were mild splenomegaly, lymph node enlargement, and hemorrhages in various organs, particularly involving the meninges and the lungs. Mediastinitis, manifested as hemorrhage or edema, affected 29% of the monkeys. Microscopically, lymphocytolysis occurred in the intrathoracic lymph nodes and spleens of all animals, and was particularly severe in the spleen and in germinal centers of lymph nodes. Hemorrhages were common in lungs, bronchial lymph nodes, meninges, gastrointestinal tract, and mediastinum. These results demonstrate that the Ames strain of B. anthracis is lethal by the inhalation route in the cynomolgus macaque. The LD(50) of the Ames strain of B. anthracis was within the expected experimental range of previously reported values in the rhesus monkey in an aerosol challenge. The gross and microscopic pathology of inhalation anthrax in the cynomolgus monkey is remarkably similar to that reported in rhesus monkeys and humans. The results of this study are important for the establishment of an alternative nonhuman primate model for evaluation of medical countermeasures against inhalational anthrax.     

Immunofluorescent and immunoelectron microscopic demonstration of primate heart muscle antigens cross-reactive with Streptococcus pyogenes.

The presence of antigens in heart muscles cross-reactive with antibodies against Streptococcus pyogenes were examined in heart samples of seven primate species (chimpanzee, rhesus macaque, stump-tailed macaque, hamadryas baboon, capuchin monkey, green monkey and patas monkey). Cross-reactive antigens were localized by means of the indirect immunofluorescent technique in nearly all samples tested. The fluorescence pattern was the same as found in the heart muscle of other mammals. Using the indirect immunoferritin technique the substructural distribution of the antigens was investigated in heart muscle of rhesus macaque and hamadryas baboon. Beside the location on the cell surface the antigens were also demonstrated within the cell. Above all there was a strong labelling along the intercalated discs and in the sarcoplasmatic reticulum.     

Competitive radioimmunoassay to detect antibodies to herpes B virus and SA8 virus.

A monoclonal competitive radioimmunoassay (CompRIAm) which detects antibody to herpesvirus simiae (B virus) in monkey and human sera and antibody to SA8 virus in monkey sera but not antibody to herpes simplex virus in human sera is described. Of 232 serum samples from wild-caught cynomolgus monkeys, 117 serum samples were positive when tested by CompRIAm. The results were in close agreement (97.5%) with B virus neutralizing antibody results on the same sera. Sera from 97 wild-caught rhesus monkeys and 92 wild-caught baboons were also tested. The CompRIAm was able to differentiate between sera that had neutralizing antibody to B virus and SA8 virus and those that did not, although the discrimination was not as clear as that in the tests on cynomolgus monkey sera. Sequential sera from two humans with confirmed cases of B virus infection were tested by CompRIAm. B virus antibody was detected in sera from both humans. None of 237 other serum samples from blood donors and patients attending sexually transmitted disease clinics reacted in the CompRIAm.     

Differential effects of administration of a human anti-CD4 monoclonal antibody, HM6G, in nonhuman primates.

A human sequence IgGkappa anti-CD4 monoclonal antibody (mAb), HM6G, originally isolated from a human immunoglobulin transgenic mouse was specific for and bound with high binding avidity to the CD4 antigen expressed on human, chimpanzee, and cynomolgus monkey T cells. Prior to testing this mAb in human clinical trials, a number of preclinical primate studies were performed. In chimpanzees, HM6G did not deplete circulating CD4(+) T cells and was cleared in a dose-dependent manner. In contrast, this mAb administered to cynomolgus monkeys depleted CD4(+) T cells (albeit only at high doses) and its clearance, which had reached saturation even at very low doses, was much slower. These differences were most likely due to the additional and rather substantial expression of the CD4 antigen on chimpanzee monocytes. In monkeys, the T cell depletion was mitigated by infusing the mAb over 30 min or longer (as opposed to 30 s) while only slightly altering the clearance. As expected, the human mAb did not induce an immune response in chimpanzees, although it did induce a low titer response in monkeys. These disparate pharmacokinetic and pharmacodynamic results suggest prudence when extrapolating results obtained in nonhuman models to humans.     

Functional characterization of non-human primate erythrocyte immune adherence receptors: implications for the uptake of immune complexes by the cells of the mononuclear phagocytic system.

Erythrocytes from primates express an immune adherence (IA) receptor that binds complement-opsonized immune complexes (IC) both in vivo and in vitro. We have analyzed the immunochemical and functional properties of the IA receptor from erythrocytes from species that have been used for in vivo IC clearance studies and have compared these properties to the human IA receptor (which is called complement receptor type 1, CR1). Erythrocytes from all species (chimpanzee, baboon, rhesus and cynomolgus monkey) bind antibody/double-stranded DNA IC when opsonized with autologous complement. However, IC which are bound to chimpanzee erythrocytes are not released upon addition of chimpanzee serum (which contains factor I activity), while IC bound to baboon erythrocytes and human erythrocytes are released upon addition of autologous serum. Anti-human CR1 monoclonal antibodies (mAb) E11 and HB8592 bind to erythrocytes from all species examined and the number of mAb epitopes per erythrocyte correlated with the number of IC that could bind to the erythrocyte under saturating conditions. However, a number of interesting differences between the species are observed with other mAb. The anti-CR1 mAb 1B4 and 3D9, which block recognition of ligand by CR1, did not bind to chimpanzee erythrocytes and bound partially to rhesus and cynomolgus monkey erythrocytes. In addition, the ability of autologous serum to induce release of erythrocyte-bound IC correlates with the presence of these epitopes. These findings, taken in context with previous clearance studies, suggest that serum-mediated release may not be required for the rapid transfer of the IC from the erythrocyte to the mononuclear phagocytic system.     

Method validation and reference range values for a peripheral blood immunophenotyping assay in non-human primates

A peripheral blood immunophenotyping assay was developed and validated for determination of total T-lymphocytes, helper T-lymphocytes, cytotoxic T-lymphocytes, B-lymphocytes, and natural killer cells in cynomolgus monkeys. Validation parameters included assessment of precision, linearity, antibody optimization, stability of peripheral blood samples, and stability of fixed immunophenotyping samples. Total lymphocyte populations were determined using a heterogeneous lymphocyte gating strategy consisting of CD45 fluorescent staining and side-scatter demarcation. Relative lymphocyte subset values were determined using antigen-specific gating strategies. Absolute subset concentrations for each lymphocyte subset were subsequently determined using a dual-platform methodology wherein relative lymphocyte subset values (via flow cytometry analyses) were multiplied by the absolute total lymphocyte (via hematology analyses) values. Reference ranges are presented for cynomolgus monkey, rhesus monkey, and baboon. Additional 1-year longitudinal immunophenotyping values are presented for the cynomolgus monkey. The method validation and reference ranges presented in this research provide a robust analytical methodology for determination of peripheral blood lymphocyte subsets in various non-human primate species.     

Anti-Interleukin-6 Antibodies in Normal Human Serum

High-avidity IgG antibodies to the cytokine interleukin-6 (IL-6) were found in sera of apparently healthy adult individuals. These antibodies specifically interfered with an ELISA (enzyme-linked immunosorbent assay) for IL-6 in which specific polyclonal rabbit antibodies to human recombinant IL-6 (rIL-6) were used. Furthermore, using precipitation of 125I-rIL-6 with rabbit antibodies to human immunoglobulins (Ig), the sera of 7 out of 68 Danish blood donors were found to contain specific antibodies in substantial amounts. Judged by ELISA interference, gel filtration of sera incubated with 125I-rIL-6 and second antibody precipitation of 125I-rIL-6, IgG seemed to be the dominant IL-6 binding protein in these normal sera. Using specific antibodies to human Ig light chains, it was found that the anti-IL-6 antibodies were of polyclonal origin. Moreover, there are at least two epitopes on the IL-6 molecule, because more than one IgG bound to some IL-6 molecules at the same time. The anti-IL-6 antibodies did not cross-react with a number of other human recombinant-derived and native cytokines. The antibodies recognized native as well as rIL-6, but preferentially monomeric IL-6.     

Production of immunoglobulin A protease by Streptococcus pneumoniae from animals.

Human isolates of Streptococcus pneumoniae tested by traditional immunochemical methods produce a protease that cleaves human immunoglobulin A1 (IgA1) into Fab and Fc fragments. The protease may be an important virulence factor, but studies of its pathogenetic significance have been hampered by lack of a suitable animal model. Since S. pneumoniae is a respiratory pathogen for several species of animals, we sought to determine whether isolates of this organism from animals with pneumococcal infection, including fatal diplococcal pneumonia, produced an IgA protease. Isolates from six animal species including the mouse, rat, dog, guinea pig, rhesus monkey, and chimpanzee were tested for protease activity against IgA preparations from the mouse, rat, dog, guinea pig, rabbit, rhesus and cynomolgus monkeys, gorilla, and human. Cleavage of IgA was demonstrated by the appearance of Fc fragments in Western blots (immunoblots) treated with specific antisera. All these isolates except that from the guinea pig produced a protease that cleaved IgA of human, rhesus monkey, and gorilla origin. Cleavage was inhibited by 5 mM EDTA. IgA cleavage from the other species could not be demonstrated. Although S. pneumoniae can colonize the respiratory tracts of several animal species, it is a significant pathogen principally of humans and some other primates. Our data suggest that some species of nonhuman primates including the rhesus monkey could be suitable for experimental studies on the significance of IgA protease in the pathogenesis of pneumococcal disease.     

Genomic DNA insertions and deletions occur frequently between humans and nonhuman primates

Comparative DNA sequence studies between humans and nonhuman primates will be important for understanding the genetic basis of the phenotypic differences between these species. Here we compare approximately 27 Mb of human chromosome 21 with chimpanzee DNA sequences identifying 57 genomic rearrangements (deletions and insertions ranging in size from 0.2 to 8.0 kb) between the two species. These rearrangements are distributed along the entire length of chromosome 21, with approximately 35% found in genomic intervals encoding genes (genic intervals), and have occurred in the genomes of both humans and chimpanzees. Comparison of approximately 9 Mb of human chromosome 21 with orangutan, rhesus macaque, and woolly monkey DNA sequences identified a combined total of 114 genomic rearrangements between humans and nonhuman primates. Analysis of these rearrangements revealed that they are randomly distributed with respect to genic and nongenic intervals and identified one deletion that has likely resulted in the inactivation of a gene (beta1,3-galactosyltransferase) in the woolly monkey. Our data show that genomic rearrangements have occurred frequently during primate genome evolution and significantly contribute to the DNA differences between these species. These DNA rearrangements are commonly found in genic intervals, and thus provide natural starting points for focused investigations of qualitative and quantitative gene expression differences between humans and other primates.     

ELISpot and ELISA analysis of spontaneous,mitogen-induced and antigen-specific cytokine production in cynomolgus and rhesus macaques

Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-gamma were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals.     

Application to immunoglobulin M capture hemadherence assays of hemagglutination of monkey erythrocytes by native and recombinant human parvovirus B19 antigens.

Human parvovirus B19 recently was shown to agglutinate baboon and human erythrocytes. We have now demonstrated that both recombinant and native B19 antigens agglutinate rhesus, cynomolgus, and Saimiri monkey erythrocytes. Using cynomolgus erythrocytes and the recombinant antigen, we developed an immunoglobulin M (IgM) antibody capture hemadherence test (MACHAT) for the detection of specific B19 IgM antibodies in human sera. The results obtained with MACHAT were compared with those obtained with an IgM capture enzyme immunoassay (MACEIA) employing the native antigen routinely used in our laboratory. For 229 patient serum samples, we found 96% agreement between the results of the two assays. There was some evidence that MACHAT was slightly more sensitive than MACEIA. Our results add to the range of erythrocytes that can be agglutinated by B19 virus and show that native as well as recombinant antigens may be used in MACHAT.     

Cloning, expression, and functional characterization of cynomolgus monkey (Macaca fascicularis) CC chemokine receptor 1

The CC chemokine receptor 1 (CCR1) has emerged as a relevant factor contributing to inflammatory diseases such as allergic asthma. Commonly used animal models of allergic airway inflammation, especially murine models, have certain limitations. The elaborate, nonhuman, primate models of asthma display the highest comparability with the situation in humans. These models play an important role in the understanding of the pathogenesis of asthma. To improve the understanding in cynomolgus monkey models, we identified and characterized CCR1 in this nonhuman primate. Initially, we cloned the cynomolgus monkey CCR1 (cCCR1) gene, and the sequence analysis revealed high homology at the nucleotide (92%) and amino acid (88.4%) levels with its human counterpart. Human embryonic kidney 293 cells were stably transfected with cCCR1 and used in functional assays. Among those CCR1 ligands tested, CCL14(9-74) was most potent in the induction of intracellular Ca2+ fluxes as observed for human CCR1 (hCCR1). Complete cross-desensitization could be achieved between CCL14(9-74) and CCL15. However, CCL3 could not fully abrogate the response to the potent ligand CCL14(9-74). Competition-binding studies with radiolabeled CCL3 concordantly showed that CCL14(9-74) has a higher affinity to cCCR1 than hCCL3. Moreover, differential tissue-specific expression of cCCR1 was investigated by real-time quantitative polymerase chain reaction, displaying the highest levels in spleen. This study adds basic information needed for the evaluation of the role of CCR1 in the pathophysiology of asthma using the highly relevant cynomolgus monkey model and in addition, aids in the preclinical evaluation of potential novel drugs targeting CCR1.     

Pregnancy zone protein analogue in pregnant and non-pregnant primates, and its decrease during pregnancy in some monkey species.

Rabbit antiserum to human pregnancy zone protein (PZP) cross-reacted with analogous proteins in several species of primates. The chimpanzee PZP showed reactions of identity with human PZP, while the PZP analogue in the orangutan, in four species of old world monkeys (pig-tailed, rhesus, cynomolgus and stump-tailed) and in a species of new world monkey (squirrel) showed equivalent reactions of partial identity with human PZP. In the chimpanzee and orangutan, the PZP analogue was present in low concentrations in non-pregnant animals, but as in the human, it increased quite appreciably during gestation. In the chimpanzee, this increase in pregnancy was about four-fold greater than in the human. In sharp contrast, in the old and new world monkeys, the PZP analogue was present in much higher concentrations in non-pregnant animals than it is in humans. In addition, during pregnancy the PZP analogue in these monkey species actually decreased during pregnancy. In the few cases studied, normal levels were regained about 1 month after delivery. A normal plasma protein, alpha2-macroglobin, was also studied in these primate species, because this protein shares some characteristics with PZP. Analogous alpha2-macroglobulin serum proteins were found in all the primates tested, but the observed gel diffusion identity patterns suggested that this protein was phylogenetically older than PZP. alpha2-macroglobulin increased slightly during human pregnancy, but in all the other primates studied, the alpha-macroglobulin analogue was either unchanged or slightly decreased during gestation.     

IL-13 blockade reduces lung inflammation after Ascaris suum challenge in cynomolgus monkeys

BACKGROUND: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. OBJECTIVE: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. METHODS: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. RESULTS: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals. CONCLUSION: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. CLINICAL IMPLICATIONS: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.     

Species-dependent serum interference in a sandwich ELISA for Apo2L/TRAIL

To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different. In order to resolve the observed serum effect, studies were performed in which salts, detergents, and blocking proteins were added to the sample diluent, and optimized sample diluents that eliminated serum interference were developed for mouse, cynomolgus monkey, and chimpanzee serum. These buffers consisted of a base assay diluent (PBS/0.5% BSA/0.05% Tween-20/10 ppm ProClin 300) supplemented with: NaCl (mouse serum); NaCl, EDTA, CHAPS, bovine gamma globulin (BGG), and human IgG (cynomolgus monkey serum); and NaCl and EDTA (chimpanzee serum). Full characterization studies were performed for the "buffer" ELISA run in base assay diluent (intended for non-serum samples) as well as the assays optimized for mouse serum and cynomolgus monkey serum. Precision, accuracy, linearity, and specificity were found to be satisfactory. With the availability of a rabbit polyclonal antibody against Apo2L/TRAIL, a new pAb/mAb ELISA was developed. This assay was not only more sensitive by > or =6-fold, but it was also much less subject to serum interference.     

Preclinical safety of recombinant human interleukin-18

Recombinant human interleukin-18 (rHuIL-18) is currently in clinical trials for treatment of cancer. This report presents results of preclinical toxicity studies with rHuIL-18 in cynomolgus monkeys and recombinant murine IL-18 (rMuIL-18) in mice. The rHuIL-18 was administered intravenously in 1 or 2 different 5-day cycles at doses 0.3 to 75 mg/kg/day in monkeys. Decreases in red cell mass, neutrophil, and platelet counts, increases in monocyte and large unstained cell counts, and lymphoid hyperplasia in spleen and lymph nodes were mild, reversible, and likely related to the pharmacologic activity of IL-18. The only toxic effect was protein cast nephropathy, secondary to coprecipitation of administered IL-18 and Tamm-Horsfall protein in the distal nephron, that only occurred at 75 mg/kg/day. Other adverse effects of rHuIL-18 were related to strong immunogenicity in monkeys and were manifest only during a second dosing cycle. The rMuIL-18, at similar dosing levels and cycles in mice, resulted in reduced red cell mass, increased white blood cell counts, spleen and lymph node hyperplasia, and mild, reversible changes in intestine, liver, and lungs. Protein cast nephropathy occurred in mice at doses > or = 30 mg/kg/day. In conclusion, preclinical safety studies showed that rIL-18 was well tolerated at pharmacologically active doses in both monkeys and mice.     

Rhesus macaque and chimpanzee DC-SIGN act as HIV/SIV gp120 trans-receptors, similar to human DC-SIGN

Dendritic cells (DC) have been implicated in the pathogenesis of both human and simian immunodeficiency viruses (HIV and SIV, respectively). The DC-specific HIV-1 trans-receptor DC-SIGN is thought to be essential for viral dissemination by DC. Abundant expression in lymphoid tissues also implies a function for DC-SIGN in chronic HIV-1 infections, in facilitating persistent infection of T cells. We have therefore isolated the rhesus macaque and chimpanzee homologues of DC-SIGN to investigate their function in a primate model. Both rhesus macaque and chimpanzee DC-SIGN are highly similar to the human homologue. Three monoclonal antibodies against human DC-SIGN, AZN-D1, -D2 and -D3, cross-react with rhesus macaque DC-SIGN, whereas AZN-D2 does not cross-react with chimpanzee DC-SIGN. The primate homologues are abundantly expressed in lymphoid tissues such as lymph nodes, as well as in mucosal tissues involved in sexual transmission of HIV-1, and are functionally similar to human DC-SIGN. They have a high affinity for the immunological ligands of DC-SIGN: ICAM-2 and -3. Moreover, both homologues bind the HIV-1 envelope glycoprotein gp120 and therefore can act as a HIV-1 trans-receptor in the same way as human DC-SIGN. These data demonstrate that primate models are suitable to further dissect the role of DC-SIGN in the transmission and pathogenesis of infection with immunodeficiency viruses.     

Threat to Humans from Virus Infections of Non-human Primates

Several hundred distinct non human primate species are recognised, and they are likely to harbour a similar range of viruses to humans. Simians such as cynomolgus and rhesus macaques, African green monkeys, and marmosets are widely used for biomedical research, but despite this extensive close contact very few simian viruses have been shown to pose a threat of infection or illness to humans. Herpesvirus Simiae is the best recognised zoonotic hazard of simians. It is an alphaherpes virus of Asiatic macaques, which causes a mild or subclinical primary infection followed by latency in its natural host. It can be acquired by humans following a bite and causes an ascending meningoencephalitis. Less than 40 human cases have been described and the mortality rate in untreated human infections is 70%. The infection is treatable with acyclovir and extensive guidelines for managing simians and potential exposures have been developed. Ebola virus and Marburg virus have caused epizootics in cynomolgus macaques and vervet monkeys respectively, which have resulted in human infection and fatalities. However, non human primates are unlikely to be their natural host. More recently simian immunodeficiency virus and simian foamy virus have infected researchers, but infection has not been linked to illness. Simian viruses also pose a direct threat to humans through the use of primary monkey tissue cultures in laboratory work and vaccine manufacture, indeed a significant exposure of the human population occurred when cells contaminated with SV40 a polyomavirus of rhesus monkeys were used for polio vaccine production. New medical interventions such as xenotransplantation using primate organs pose a potential risk which requires careful assessment. © 1997 by John Wiley & Sons Ltd.     

Comparative FISH mapping of the ancestral fusion point of human chromosome 2

It is known that human chromosome 2 originated from the fusion of two ancestral primate chromosomes. This has been confirmed by chromosome banding and fluorescence in-situ hybridization (FISH) with human chromosome-2-specific DNA libraries. In this study, the order of 38 cosmid clones derived from the human chromosome region 2q12-q14 was exactly determined by high-resolution FISH in human chromosome 2 and its homologous chromosomes in chimpanzees (Pan trogrodydes, 2n=48) and cynomolgus monkeys (Macaca fascicularis, 2n=42). This region includes the telomere-to-telomere fusion point of two ancestral ape-type chromosomes. As a result of comparative mapping, human chromosome region 2q12-q14 was found to correspond to the short arms of chimpanzee chromosomes 12 and 13 and cynomolgus monkey chromosomes 9 and 15. It is noted that no difference was detected in the relative order of the cosmid clones between human and chimpanzee chromosomes. This suggests that two ancestral ape-type chromosomes fused tandemly at telomeres to form human chromosome 2, and the genomic organization of this region is thought to be considerably conserved. In the cynomolgus monkey, however, the order of clones in each homologue was inverted. In addition to cosmid mapping, two chromosome-2-specific yeast artificial chromosome (YAC) clones containing the fusion point were identified by FISH. This revised version was published online in July 2006 with corrections to the Cover Date.