Rat-specific decreases in platelet count caused by a humanized monoclonal antibody against sclerostin.

LY2541546 is a humanized monoclonal antibody (IgG(4)) that has been optimized for neutralizing activity against sclerostin. In 5-week and 6-month nonclinical safety studies in rats, LY2541546 caused dose-dependent reversible decreases in platelet counts accompanied by accelerated platelet production, increased megakaryocytes, and altered megakaryocyte morphology. These treatment-related effects resulted in altered primary hemostasis as manifested by prolonged bleeding after phlebotomy or incidental toenail break. In some cases, the defects in hemostasis were sufficient to result in death of the affected rats. There was no evidence in rats of general bone marrow suppression or processes (e.g., disseminated intravascular coagulopathy) that may result in thrombocytopenia. Cynomolgus monkeys given LY2541546 for 5 weeks or 9 months had no changes in platelet count or megakaryocytes. In vitro cross-reactivity studies in rats, cynomolgus monkeys, and humans revealed LY2541546-bound rat but not cynomolgus monkey or human platelets and megakaryocytes. These data taken together demonstrated that the platelet and megakaryocyte effects in rats had a species-specific pathogenesis which likely involved LY2541546 binding of a rat-specific antigen on the surface of platelets and megakaryocytes resulting in the increased clearance of platelets and megakaryocyte hyperplasia. The species-specific nature of these reversible toxicological findings combined with the ease of clinical monitoring using standard hematology enabled the safe initiation of clinical studies in human volunteers.

Interstitial myocardial fibrosis in a captive chimpanzee (Pan troglodytes) population.

The clinical and necropsy records of 36 (25 male and 11 female) chimpanzees age 10 to 40 y old that died over a 6-y period (2001 to 2006) were reviewed. All animals had annual physical exams that included electrocardiograms and serial blood pressures. Nine of the 36 animals had a complete cardiac evaluation by a board certified veterinary cardiologist, and 7 of the 36 animals (19%) were diagnosed with some form of cardiomyopathy. Systemic hypertension was noted in 3 cases. Cardiac arrhythmias (ventricular ectopy) were seen in 15 (12 male and 3 female) of the 36 animals (42%). Sudden cardiac death (SCD) occurred in 13 (11 male and 2 female) chimps (36%) and was the leading cause of death (n = 13), followed by renal failure (n = 9) and septicemia (n = 3). Histologic examination of the hearts revealed interstitial myocardial fibrosis (IMF) in 29 chimpanzees (81%), and all of the animals that died suddenly due to cardiac causes had IMF to varying degrees. More data will be needed to identify the possible causes of IMF in captive chimpanzees, and IMF may be associated with arrhythmias and SCD in these animals.

Serial electrophysiologic studies in rhesus monkeys with Krabbe disease.

Krabbe disease is a progressive leukodystrophy that results in demyelination in the central and peripheral nervous systems in humans. It has been described in a number of mammalian species including the rhesus monkey. We performed serial nerve conduction studies beginning within the first 2 months of life in four homozygous, two heterozygous, and two normal rhesus monkeys (Macaca mulatta) to characterize the peripheral neuropathy. Mean conduction velocities of the median, ulnar, and tibial nerves were significantly slower in the affected than unaffected monkeys at all ages (P < 0.0001). The conduction velocity differences became more apparent between the affected and unaffected as the monkeys aged. When compared to the unaffected monkeys, the serial conduction velocities suggested occurrence of dysmyelination followed by demyelination in the affected monkeys. These observations provide further insight into the disease process and suggest an early window of opportunity for treating Krabbe disease.

Direct inoculation of simian immunodeficiency virus from sooty mangabeys in black mangabeys (Lophocebus aterrimus): first evidence of AIDS in a heterologous African species and different pathologic outcomes of experimental infection.

A unique opportunity for the study of the role of serial passage and cross-species transmission was offered by a series of experiments carried out at the Tulane National Primate Research Center in 1990. To develop an animal model for leprosy, three black mangabeys (BkMs) (Lophocebus aterrimus) were inoculated with lepromatous tissue that had been serially passaged in four sooty mangabeys (SMs) (Cercocebus atys). All three BkMs became infected with simian immunodeficiency virus from SMs (SIVsm) by day 30 postinoculation (p.i.) with lepromatous tissue. One (BkMG140) died 2 years p.i. from causes unrelated to SIV, one (BkMG139) survived for 10 years, whereas the third (BkMG138) was euthanized with AIDS after 5 years. Histopathology revealed a high number of giant cells in tissues from BkMG138, but no SIV-related lesions were found in the remaining two BkMs. Four-color immunofluorescence revealed high levels of SIVsm associated with both giant cells and T lymphocytes in BkMG138 and no detectable SIV in the remaining two. Serum viral load (VL) showed a significant increase (>1 log) during the late stage of the disease in BkMG138, as opposed to a continuous decline in VL in the remaining two BkMs. With the progression to AIDS, neopterin levels increased in BkMG138. This study took on new significance when phylogenetic analysis unexpectedly showed that all four serially inoculated SMs were infected with different SIVsm lineages prior to the beginning of the experiment. Furthermore, the strain infecting the BkMs originated from the last SM in the series. Therefore, the virus infecting BkMs has not been serially passaged. In conclusion, we present the first compelling evidence that direct cross-species transmission of SIV may induce AIDS in heterologous African nonhuman primate (NHP) species. The results showed that cross-species-transmitted SIVsm was well controlled in two of three BkMs for 2 and 10 years, respectively. Finally, this case of AIDS in an African monkey suggests that the dogma of SIV nonpathogenicity in African NHP hosts should be reconsidered.

Retrospective analysis of clinical and laboratory factors associated with lymphoma in simian AIDS.

A nonhuman primate model for AIDS-associated Non-Hodgkin's lymphoma (AIDS-NHL) has been described in which animals inoculated with simian immunodeficiency virus (SIV) develop simian AIDS (SAIDS) and SAIDS-NHL. The objective of the present study was to describe statistically the major trends observed in clinical and laboratory data collected longitudinally on a large cohort of nonhuman primates that developed SAIDS-NHL. Clinical and laboratory data were collected longitudinally on each animal from the time of SIV infection throughout progression to lymphoma. Data were analyzed retrospectively with regard to species, gender, age at SIV inoculation, survival, cause of death, CD4+ T-cell and B-cell counts, SIV antigenemia, persistent lymphoid hyperplasia and lymphocryptovirus infection. Median survival time (354 days: 95% CI 309-388) was not related to gender, age at SIV inoculation, cause of death, or RhLCV infection. Survival was not related to CD4+ T-cell count at the time of SIV infection (P = 0.5531), but increased survival was significantly related to a slower rate of CD4+ T-cell decline (P = 0.0256). A B-cell expansion was observed at the midpoint of disease. A steep rise in SIV antigenemia was detected in the first 21 days of infection followed by a rapid decline. This pattern did not occur in animals inoculated with SIV as infants or yearlings. Of 45 cases, 9 exhibited marked, persistent lymphoid hyperplasia. These results describe trends identified in clinical and laboratory factors associated with SAIDS-NHL in the largest collection of such samples in the world. The results contribute to an understanding of the etiology of SAIDS-NHL and to the future development of useful predictors of SAIDS- or AIDS-related lymphoma.

Antibody responses and protection from pyelonephritis following vaccination with purified Escherichia coli PapDG protein.

PURPOSE: A critical early step in the establishment of Escherichia coli pyelonephritis is bacterial attachment via the tip protein of P fimbriae. This adhesin, PapG, binds to glycolipid receptors present on vaginal and kidney epithelial surfaces. In this study we investigated the efficacy of vaccination with purified PapDG protein complex in preventing pyelonephritis caused by E. coli. MATERIALS AND METHODS: Mature cynomolgus monkeys were intraperitoneally vaccinated with 100 microg purified PapDG protein. Following 3 identical boosters serum antibody titers to PapDG were measured by enzyme-linked immunosorbent assay. Vaccinated and unvaccinated animals were urethrally inoculated with 1 x 10 cfu of E. coli strain DS17, which was isolated from a child with acute pyelonephritis. The infection course was monitored by catheterized urine cultures, and by histological examination of the kidneys, bladder and kidney tissue culture 28 days after infection. RESULTS: Intraperitoneal administration of purified PapDG vaccine resulted in the development of specific antibody responses in cynomolgus monkeys. In contrast to control monkeys, vaccinated monkeys did not show histological evidence of pyelonephritis after subsequent urethral challenge with pyelonephritogenic E. coli expressing P fimbriae. CONCLUSIONS: Purified PapDG is a tractable vaccine candidate that in our small study demonstrated the ability to elicit adequate serum antibody levels to prevent E. coli mediated pyelonephritis.

A brief history of the discovery of natural simian immunodeficiency virus (SIV) infections in captive sooty mangabey monkeys.

Experimental leprosy studies using Mycobacterium leprae inoculum isolated from a sooty mangabey monkey (SMM) resulted in the accidental discovery that SMM's asymptomatically carry simian immunodeficiency virus (SIV) that is pathogenic in macaques. We showed that the SMM virus, SIVDelta, was antigenically related to SIVmac, which had been identified in macaques, and to the human immunodeficiency virus (HIV). Similar asymptomatic natural SIV infections had been reported in African green monkeys (AGM). Our results together with observations of others led us to propose that both SIVmac and SIVDelta originated in SMM and that SIV emerged in humans as a result of early African nonhuman primate SIV trans-species infections in humans.

A preliminary evaluation of recombinant adeno-associated virus biodistribution in rhesus monkeys after intrahepatic inoculation in utero.

The ability to deliver genes to fetuses in utero may prove crucial for those genetic diseases that are associated with severe fetal morbidity and for which there is no effective postnatal therapy. In utero therapy may be especially useful in diseases that affect the central nervous system because the immature blood-brain barrier may facilitate gene delivery to neural target cells. We investigated whether in utero inoculation of recombinant adeno-associated virus (rAAV) into rhesus monkey fetuses would be a useful method of gene delivery, especially to the central nervous system. When the monkeys were sacrificed after birth, we found vector genomes distributed in many tissues, including the brain and peripheral blood. Pericapillary astrocytes expressing transgene products were detected by immunohistochemistry. In addition, we occasionally found vector genomes in the maternal blood. No adverse clinical or pathologic effects were observed in the inoculated monkeys. We concluded that (1) in utero intrahepatic inoculation of rAAV is a potentially safe and useful method of delivering genes to many fetal tissues; (2) astrocytes may be the cell type most easily targeted in the central nervous system (CNS) after systemic administration; and (3) the potential of inadvertent gene transfer to the mother must be considered.

Liver regeneration investigated in a non-human primate model (Macaca mulatta).

BACKGROUND/AIMS: An adequate model to study liver regeneration in humans is presently unavailable. We explored the feasibility of studying liver regeneration in a genetically similar species to man, the non-human primate Rhesus macaque. METHODS: Five animals were studied; two underwent 60% hepatectomy, one underwent 30% hepatectomy, and cholecystectomy alone was performed on two animals. Laparoscopic-guided or open liver biopsies were performed on days 1, 2, 7, 14, 21, 30 and 60 following all surgeries. Liver regeneration was evaluated by measuring Ki-67, proliferating cell nuclear antigen expression and mitotic index, calculating changes in the surface area of the liver remnant and assessing intrahepatic production of cytokines. RESULTS: Significant liver regeneration was induced in the animals that underwent 60% hepatectomy, peaking between days 21-30 postoperatively. Regeneration was minimal in all other animals studied. Cytokine production followed a similar pattern. Maximal liver regeneration correlated with restoration of surface area in the liver remnant. CONCLUSIONS: Sixty percent hepatectomy in a non-human primate model induced significant liver regeneration, maximizing 21-30 days following partial hepatectomy, suggesting a significant interspecies difference when compared to a rodent hepatectomy model. A partial hepatectomy model in Rhesus macaques may allow further characterization of liver regeneration in a species closer to humans.

Use of perflubron to enhance lung gene expression: safety and initial efficacy studies in non-human primates.

Use of perflubron (LiquiVent) and other perfluorochemical liquids during intratracheal administration of adenovirus and AAV vectors has been shown to improve total gene expression as well as distribution of expression throughout lungs of spontaneously breathing rodents. To determine if this method could be safely and easily extended to non-human primates, we carried out a pilot investigation in six spontaneously breathing rhesus macaques. Two animals received bronchoscopic administration of recombinant adenovirus vector (type 5 E1-deleted AdCMVlacZ, 4.6 x 10(10) plaque forming units/animal), two animals received vector followed by instillation of perflubron, and two animals received perflubron alone. Instillation of perflubron was well tolerated by the animals and, once recovered from anesthesia, all animals behaved and fed normally until lung harvest. Serial X-rays demonstrated that the perflubron had cleared from lungs of three animals by 48 hours after administration; the fourth animal had a small amount of residual perflubron. Apart from a mild elevation in hepatocellular enzymes, no significant abnormality was noted in complete blood count or serum electrolytes and chemistries. In animals receiving either vector alone or vector with perflubron, in situ beta-galactosidase expression was observed in a variety of cells including large airway, bronchiolar, and alveolar epithelial cells. In summary, use of perflubron was well tolerated in spontaneously breathing macaques. Further studies in larger numbers of animals will help assess the potential efficacy of perflubron for enhancing gene expression and elucidate effects on local and systemic inflammatory responses.

Antileprosy protective vaccination of sooty mangabey monkeys with BCG or BCG plus heat-killed mycobacterium leprae: immunologic observations

Groups of sooty mangabey monkeys (SMM) were vaccinated and boosted with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or BCG + low-dose (LD) or high-dose (HD) heat-killed M. leprae (HKML), or were unvaccinated. Prior to and following vaccination-boosting and subsequent M. leprae (ML) challenge, these and unvaccinated, unchallenged control monkeys were immunologically observed longitudinally for approximately 3 years. SMM [multibacillary (MB) leprosy-prone as a species] were not protected clinically by BCG or BCG + HKML, although the disease progress was slowed by vaccination with BCG alone. The longitudinal immune response profiles to BCG or BCG + HKML in SMM showed that: 1) vaccination with BCG or BCG + HKML initially stimulated significant in vitro blood mononuclear cell blastogenic responses to ML antigens, which returned to baseline post-boosting and post-live ML challenge; 2) BCG + LD HKML-vaccinated groups gave the largest blastongenic response (SI = 23) followed by the BCG + HD HKML group (SI = 14.5) and by the BCG-only vaccinated group (SI = 3.6); 3) significantly diminished numbers of blood CD4+ (helper) and CD4+CD29+ (helper-inducer) T-cell subsets were observed longitudinally in all ML-challenged groups compared to controls regardless of whether they had been vaccinated or not; 4) CD8+ (suppressor) T-cell numbers remained longitudinally constant, on average, in all ML-challenged groups (vaccinated or not) compared to controls; 5) there was a significant decrease in the CD4+:CD8+ ratio over time in all ML-challenged groups (vaccinated or not); 6) vaccination with BCG or BCG + LD or HD HKML resulted in significantly increased numbers of CD4+CD45RA+ (suppressor-inducer) T cells longitudinally compared to the unvaccinated, ML-challenged control group; and 7) over time, vaccination with BCG + HKML followed by live ML-challenge produced higher IGM:IgG antiphenolic glycolipid-I (PGL-I) serum antibody response ratios than BCG-only vaccinated, ML-challenged monkeys or unvaccinated, ML-challenged SMM, consistent with prior observations that IgG anti-PGL-I responses correlate with resistance to and protection from clinical leprosy and IgM anti-PGL-I responses correlate with increased susceptibility.

Antileprosy protective vaccination of rhesus monkeys with BCG or BCG plus heat-killed mycobacterium leprae: immunologic observations

Groups of rhesus monkeys were vaccinated and boosted with Mycobacterium bovis bacillus Calmette Guerin (BCG) or BCG plus low-dose (LD) or high-dose (HD) heat-killed M. leprae (HKML), or were unvaccinated. Prior to and following vaccination-boosting and subsequent M. leprae (ML) challenge, these and unvaccinated, unchallenged control monkeys were observed longitudinally for approximately 3 years. Vaccination with BCG plus HKML initially stimulated significant in vitro blood mononuclear cell blastogenic responses to lepromin, which returned to baseline post-boosting and post-live-ML-challenge, minimally reappearing significantly 2 years post-ML-challenge. Vaccination with BCG failed to stimulated positive blastogenic responses to lepromin before ML-challenge but small, marginally positive, intermittent responses were seen post-ML-challenge. Compared to the unvaccinated ML-challenged group, significant increases in the numbers of blood CD4+ and CD8+ T-cell subsets and an increased CD4+:CD8+ ratio were observed in both BCG plus HKML-vaccinated, ML-challenged groups, but not in the BCG-only-vaccinated, ML-challenged group. CD4+CD29+ and CD4+CD45RA+ subset numbers increased significantly over time in only the BCG plus LD HKML-vaccinated, ML-challenged group. Compared to unvaccinated, ML-challenged groups, vaccination with BCG or BCG plus HKML followed by ML-challenge produced lower IgM:IgG antiphenolic glycolipid-I (PGL-I) serum antibody ratios and protected rhesus monkeys from clinical leprosy, consistent with prior observations that low IgM:IgG anti-PGL-I responses correlated with resistance to and protection from leprosy.

Specific passage of simian immunodeficiency virus from end-stage disease results in accelerated progression to AIDS in rhesus macaques.

To determine whether passage of late-stage variants of simian immunodeficiency virus (SIV) would lead to a more virulent infection and rapid disease progression, a study was designed to examine the effects of selective transmission of SIV from late-stage cases of AIDS in Macaca mulatta. In a uniform group of 10 age-matched animals from the same genetic breeding stock infected with SIV(B670), it took 7 months before one of the ten animals developed AIDS. Passage of virus taken from this animal immediately prior to death resulted in death of the recipient due to AIDS within 4 months. Again, subsequent passage of virus taken late in disease resulted in an accelerated disease course, with AIDS developing within 2.5 and 1.8 months in two recipients. The fourth passage of virus taken late in disease from the most rapid progressor (1.8 months) resulted in AIDS developing in this recipient within 1 month of infection. During each consecutive passage in vivo, the loss of memory T cells became more acute. Evidence that the virus became more virulent with selective passage of late-stage variants was provided by the markedly increased levels of both plasma antigen and viral RNA. Subsequent in vivo passage from end-stage AIDS selected for a strain of SIV capable of causing the acute development of AIDS as rapidly as 1 month post-infection. The pathology of acute AIDS in these cases closely resembled that seen after a chronic disease course.