Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Spontaneous Reproductive Tract Lesions in Aged Captive Chimpanzees.

Chimpanzees (Pan troglodytes) have served as an important model for studies of reproductive diseases and aging-related disorders in humans. However, limited information is available about spontaneously occurring reproductive tract lesions in aging chimpanzees. In this article, the authors present histopathologic descriptions of lesions identified in the reproductive tract, including the mammary gland, of 33 female and 34 male aged chimpanzees from 3 captive populations. The most common findings in female chimpanzees were ovarian atrophy, uterine leiomyoma, adenomyosis, and endometrial atrophy. The most common findings in male chimpanzees were seminiferous tubule degeneration and lymphocytic infiltrates in the prostate gland. Other less common lesions included an ovarian granulosa cell tumor, cystic endometrial hyperplasia, an endometrial polyp, uterine artery hypertrophy and mineralization, atrophic vaginitis, mammary gland inflammation, prostatic epithelial hyperplasia, dilated seminal vesicles, a sperm granuloma, and lymphocytic infiltrates in the epididymis. The findings in this study closely mimic changes described in the reproductive tract of aged humans, with the exception of a lack of malignant changes observed in the mammary gland and prostate gland.

Spontaneous high-grade glial intramedullary tumor of the spine in a rhesus macaque (Macaca mulatta).

BACKGROUND: A 4-year-old rhesus macaque presented with acute, progressive paresis of the extremities. METHODS: A complete blood count, serum biochemical analysis, neurologic exam and necropsy were performed. RESULTS: The clinical, histopathological, and immunohistochemical findings confirmed a high-grade intramedullary glial tumor of the spinal cord that was most consistent with an ependymoma. CONCLUSIONS: We describe a case of a naturally occurring spontaneous spinal cord neoplasia in a non-human primate.

Campylobacter-induced fetal death in a rhesus monkey.

A pregnant 4-year-old rhesus monkey (Macaca mulatta) was presented with a history of acute vaginal bleeding. Physical examination revealed an open cervix. An ultrasound scan demonstrated a viable early third-trimester fetus, approximately 16 weeks of gestational age. Hematology results showed that the monkey was anemic, with a normal leukogram and Döhle bodies. A subsequent cervical culture was positive for Campylobacter fetus. The fetus died 3 days later, and a necropsy of the fetus and placenta was performed. Microscopic examination of the placenta revealed villitis, perivillitis, and deciduitis with S-shaped and gull wing-shaped bacteria. C. fetus was considered the cause of the placental lesions and fetal death; however, the pathogenesis of the infection (hematogenous vs. ascending from the maternal genital tract) was not clear. This is the first report of a Campylobacter-induced fetal death in the rhesus monkey. Because macaques can be asymptomatic carriers and Campylobacter-induced diarrhea is common, this finding has implications for breeding success in nonhuman primate breeding colonies.

Protection against chronic infection and AIDS by an HIV envelope peptide-cocktail vaccine in a pathogenic SHIV-rhesus model.

Based on our prior studies in mouse, monkey, chimpanzee, and human experimental systems, we identified six peptides encoded by highly conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope gene that selectively induce cellular immune responses in the absence of anti-viral antibody production. We tested a cocktail of the six peptides as a prototype vaccine for protection from simian human immunodeficiency virus (SHIV) infection and acquired immunodeficiency syndrome (AIDS) in a rhesus monkey model. Three monkeys were vaccinated with the peptide cocktail in Freund's adjuvant followed by autologous dendritic cells (DC) pulsed with these peptides. All the vaccinated animals exhibited significant induction of T-cell proliferation and cytotoxic T lymphocytes (CTL) responses, but no neutralizing antibodies. Two control mock-vaccinated monkeys showed no specific immune responses. Upon challenge with the pathogenic SHIV(KU-2), both the control and vaccinated monkeys were infected, but efficient clearance of virus-infected cells was observed in all the three vaccinated animals within 14 weeks. These animals also experienced a boosting of antiviral cellular immune responses after infection, and maintained antigen-specific IFN-gamma-producing cells in circulation beyond 42 weeks post-challenge. In contrast, the two mock-vaccinated monkeys had low to undetectable cellular immune responses and maintained significant levels of viral-infected cells and infectious virus in circulation. Further, in both the control monkeys plasma viremia was detectable beyond 38 weeks post-challenge indicating chronic phase infection. In one control monkey, the CD4+ cells dropped to very low levels by 2 weeks post-challenge and became undetectable by week 39 coinciding with high plasma viremia and AIDS, which included cachexia and ataxia. These results serve as proof of principle for the effectiveness of the HIV envelope peptide cocktail vaccine against chronic infection and AIDS, and support the development of multivalent peptide-based vaccine as a viable strategy to induce cell-mediated immunity (CMI) for protection against HIV and AIDS in humans.

Rhesus macaque model for ovarian cancer chemoprevention.

PURPOSE: The objective of the study reported here was to explore whether a nonhuman primate model could be developed for chemoprevention of ovarian cancer. METHODS: An initial feasibility trial was done with three monkeys to determine tolerance for these drugs and for acquisition of surgical ovarian biopsy specimens. In the study, 19 female adult Macacca mulatta (rhesus macaques) were given fenretinide (4HPR) oral contraceptive (OCP), the combination of 4HPR+OCP, or no medication for three months. Laparotomy was performed before and after drug administration, and ovarian biopsy specimens were obtained to evaluate the potential for this animal as a model for ovarian cancer chemoprevention, as well as evaluating fluorescence spectroscopy and other potential biomarkers for ovarian cancer prevention studies. RESULTS: The monkeys tolerated the drugs, surgeries, and acquisition of multiple ovarian biopsy specimens with resultant minimal morbidity. On initial data analysis, fluorescence spectroscopy was the marker that appeared the most promising. CONCLUSIONS: On the basis of results of this study, this model merits further investigation. The rhesus monkey is an excellent candidate for a nonhuman primate model for ovarian cancer chemoprevention.

Antagonism of soman-induced convulsions by midazolam, diazepam and scopolamine.

The effects of midazolam (MDZ), diazepam (DZ) and scopolamine (SCP) therapies on soman-induced electrocorticogram (ECoG) and biceps femoris electromyogram (EMG) activities and brain lesions were assessed in male rats. Animals received pyridostigmine (26 micrograms/kg, im) 30 min before soman (87.1 micrograms/kg, im) followed by therapy consisting of atropine (1.5 mg/kg) admixed with 2-PAM (25 mg/kg, im) 1 min later; MDZ (0.5 mg/kg), DZ (1.77 mg/kg) or SCP (0.43 mg/kg) was administered im at 1 min after the onset of convulsions (CVs). Typically, within 5 min after soman the ECoG profile changed to a full-blown, spike-and-dome epileptiform (SDE) pattern followed by CVs and increased amplitude of EMG activity. Treatment with SCP restored ECoG and EMG profiles by 30 min. At 2 hr after exposure only 1 animal demonstrated a slight abnormality in ECoG activity which was normal at 24 hr. Similarly, DZ and MDZ restored EcoG and EMG profiles by 30 min; however, in contrast to SCP, 83% of the animals demonstrated reappearance of SDE 2 hrs after soman. SCP therapy also enabled rats to move about in their cages by 30 min post treatment. In contrast, DZ- and MDZ-treated rats remained incapacitated as late as 2 hr post-exposure. Animals were euthanized at 24 hr, and the extent of soman-induced brain lesions was determined by light microscopic analysis. When present, brain lesions were minimal in SCP-treated rats. The mean brain lesion scores across all experimental conditions ranked as follows: soman control > MDZ > DZ > or = SCP = saline control. These observations suggest that SCP may be highly effective in severe soman intoxication.

Immunogenicity and efficacy against lethal aerosol staphylococcal enterotoxin B challenge in monkeys by intramuscular and respiratory delivery of proteosome-toxoid vaccines.

Staphylococcal enterotoxin B (SEB), a primary cause of food poisoning, is also a superantigen that can cause toxic shock after traumatic or surgical staphylococcal wound [correction of would] infections or viral influenza-associated staphylococcal superinfections or when aerosolized for use as a potential biologic warfare threat agent. Intranasal or intramuscular (i.m.) immunization with formalinized SEB toxoid formulated with meningococcal outer membrane protein proteosomes has previously been shown to be immunogenic and protective against lethal respiratory or parenteral SEB challenge in murine models of SEB intoxication. Here, it is demonstrated that immunization of nonhuman primates with the proteosome-SEB toxoid vaccine is safe, immunogenic, and protective against lethal aerosol challenge with 15 50% lethal doses of SEB. Monkeys (10 per group) were primed i.m. and given booster injections by either the i.m. or intratracheal route without adverse side effects. Anamnestic anti-SEB serum immunoglobulin G (IgG) responses were elicited in all monkeys, but strong IgA responses in sera and bronchial secretions were elicited both pre- and post-SEB challenge only in monkeys given booster injections intratracheally. The proteosome-SEB toxoid vaccine was efficacious by both routes in protecting 100% of monkeys against severe symptomatology and death from aerosolized-SEB intoxication. These data confirm the safety, immunogenicity, and efficacy in monkeys of parenteral and respiratory vaccination with the proteosome-SEB toxoid, thereby supporting clinical trials of this vaccine in humans. The safety and enhancement of both bronchial and systemic IgA and IgG responses by the proteosome vaccine delivered by a respiratory route are also encouraging for the development of mucosally delivered proteosome vaccines to protect against SEB and other toxic or infectious respiratory pathogens.

Aerosolized staphylococcal enterotoxin B-induced pulmonary lesions in rhesus monkeys (Macaca mulatta).

The pathology of aerosolized staphylococcal enterotoxin B (SEB) was studied in the nonhuman primate. Six juvenile rhesus monkeys that received multiple lethal inhaled doses of SEB developed diarrhea and vomiting within 24 hr followed by depression, dyspnea, and shock. Three of 6 animals died by 52 hr. The most striking gross lesion in all 6 monkeys was diffuse severe pulmonary edema. Histologically, edema fluid was present within the peribronchiolar, peribronchial, and perivascular interstitium, alveolar septa, and alveoli. The adventitia of pulmonary vessels was infiltrated by lymphocytes, macrophages, and fewer neutrophils. Numerous large lymphocytes with occasional mitotic figures were within pulmonary vessels, often occluding alveolar capillaries. These cells were strongly immunoreactive with monoclonal antibodies against CD3, establishing them as T cells. Ultrastructurally, endothelial cell junctions were intact, and endothelial cells and type I pneumocytes contained numerous pinocytotic vesicles. Alveolar septal interstitial spaces were expanded by edema. The mechanism of these SEB-induced pulmonary lesions was not determined. We hypothesize that cytokine production by activated T cells may have caused vascular permeability changes leading to widespread pulmonary edema and shock.

Increased susceptibility to staphylococcal enterotoxin B intoxication in mice primed with actinomycin D.

Mice (BALB/cJ, C3H/HeN, and C3H/HeJ) primed with actinomycin D became highly susceptible to lethal intoxication with staphylococcal enterotoxin B (SEB). The mice underwent toxicosis and toxic shock and died. Actinomycin D-primed C3H/HeN and C3H/HeJ mice showed equal sensitivity to SEB, suggesting that bacterial lipopolysaccharide derived from gram-negative bacteria in the gut may not be an important cofactor in intoxication. In a time course study of the illness, prominent pathological changes characterized by blood congestion and thickening of alveolar septa were seen in the lung, while blood congestion, inflammation, epithelial cell flattening, and villous blunting were seen in the small intestine. In lymphoid tissues, such as the spleen, congestion, inflammation, and lymphoid cell depletion were the major reactions. The pathological features of the mice had many similarities to those of rhesus monkeys intoxicated with intravenous SEB. The actinomycin D-primed C3H/HeJ mice are thus an ideal mouse model for studying SEB toxicosis and toxic shock.

Soman-induced morphological changes: an overview in the non-human primate.

A review of the literature was conducted to provide an overview of organophosphorus (OP)-induced morphological changes in the non-human primate. Most studies have evaluated effects of the OP nerve agent soman (pinacolyl methylphosphonofluoridate), an irreversible inhibitor of acetylcholinesterase. Soman-induced acute and chronic morphological changes have been examined. The effects of nerve agent therapy (i.e. pyridostigmine, praloxidime chloride and atropine), with and without an anticonvulsant (i.e. diazepam, midazolam), on soman-induced lesions have also been studied. Acute changes in the central nervous system of rhesus and cynomolgus monkeys exposed to soman alone or soman and therapy, without an anticonvulsant, were characterized by neuronal degeneration and necrosis and neuropil edema. The lesions were usually present in the frontal cortex, entorhinal cortex, amygdaloid complex, caudate nucleus, thalamus and hippocampus. Morphologically, these lesions resemble lesions produced by hypoxic-ischemic injury or by seizures and are similar to soman-induced changes in other laboratory animals. Nerve agent therapy supplemented with an anticonvulsant reduced or prevented soman-induced acute neural lesions. Acute changes in non-neural tissues were limited to the heart (e.g. hemorrhage, myofiber necrosis, myocarditis) and skeletal muscle (e.g. myofiber necrosis). Heart lesions in the non-human primate are similar to OP-induced heart lesions in man. The pathogenesis of the acute lesions in both the central nervous system and heart is discussed. Consistent soman-induced chronic morphological changes have not been produced in the rhesus monkey or baboon.

Virus-like particles in the liver of a patient with fulminant hepatitis and antibody to hepatitis E virus.

In earlier studies, hepatitis E virus (HEV) particles were detected in the stools of patients with enterically transmitted non-A, non-B (ENANB) hepatitis, and HEV was etiologically associated with this disease. Such particles have not been observed in the liver, however. We describe the pathological findings in the liver of a young pregnant woman from Nepal who died as a result of fulminant NANB hepatitis. IgM antibody to HEV was detected in the patient's serum by immune electron microscopy, suggesting that she was acutely infected with that virus. On light microscopic examination of the liver we observed cholestatic hepatitis with proliferation of bile ductules and pseudoglandular arrangement of hepatocytes around distended bile canaliculi. Three types of virus-like particles were detected by electron microscopy. The most frequently observed particles were in cells lining small bile ductules; they measured 32-37 nm and were enclosed by a membrane. Particles of a second type were seen in clusters in the sinusoidal cells; they were uniform in size, without a membrane, and measured about 32 nm in diameter. Particles of a third type (65 nm) were found in epithelial cells of the small bile ductules. Among the particles we detected, the 32 nm particles most closely resembled those of HEV.

Seroprevalence of Helicobacter pylori infections in Thailand.

Serologic studies in developed countries indicate that Helicobacter (formerly Campylobacter) pylori infection is uncommon until the third decade of life and achieves a peak prevalence of 50% in the seventh decade. In developing countries the epidemiology of H. pylori has not well been described. A sensitive and specific serologic assay for H. pylori infection was validated in Thai patients also studied by culture and histologic examination of biopsy specimens. The prevalence of H. pylori antibodies in persons from a rural Thai community began early (17.5% of children 5-9 years old), increased to 55% during the third decade of life, and peaked (75%) in the 30- to 49-year age group. At a Bangkok orphanage where enteric infections are hyperendemic, 74% of children 1-4 years old were seropositive. This study shows that the prevalence of H. pylori infection in Thailand is higher than in industrialized countries. The high infection rate at the orphanage suggests that person-to-person transmission of H. pylori may be occurring.

Pathologic activity of Plasmodium berghei prevented but not reversed by dexamethasone.

Dexamethasone has recently been shown to block the production of cachectin (implicated in the pathogenesis of cerebral malaria) if administered prior to endotoxin induction of mouse macrophages. Using the hamster cheek pouch-cerebral malaria model, we tested the hypothesis that dexamethasone is effective as a therapeutic agent in severe malaria if given before some yet undefined trigger point in the disease. Infected hamsters were treated with dexamethasone (0.7 mg/kg) daily on days 7-12, 4-12, or 1-12 post-challenge. When treatment was started on day 1, whole body oxygen consumption (used as a measure of erythrocyte transport to sites of diffusion) on day 12 was greater than (P less than 0.05) that of infected control animals, though the degree of anemia was no different in treated and untreated groups. Furthermore, treatment produced a reduction in monocyte accumulation, capillary malfunction, and monocyte/red blood cell aggregate formation observable in the cheek pouch in vivo and a similar reduction in monocyte presence, capillary pathologic change, and multifocal hemorrhage in the brain on postmortem. These data suggest that mediator(s), whose production can be blocked by pretreatment with dexamethasone, are involved in the pathogenesis of disease leading to death of the Plasmodium berghei infected hamster.

Deteriorating stroke model: histopathology, edema, and eicosanoid changes following spinal cord ischemia in rabbits.

Secondary motor dysfunction is often observed following ischemic episodes in the central nervous system. To study potential mechanisms of postischemic motor deterioration, we developed a rabbit spinal cord ischemia model that has characteristics similar to the clinical condition termed deteriorating stroke. In this model, 70% of the rabbits regained substantial motor function by 4 hours after complete hindlimb paralysis during lumbar spinal cord ischemia; however, over the next 20 hours motor function steadily declined to the point where only 30% of the rabbits had minimal hopping function. The role of eicosanoids in spinal cord ischemia was studied by radioimmunoassay of several prostaglandins (6-keto-PGF1 alpha, PGE2, and TxB2) in the spinal cord. After 5 minutes of reperfusion, TxB2 levels were markedly elevated (p less than 0.05) while 6-keto-PGF1 alpha levels did not change. The TxB2:6-keto-PGF1 alpha ratio was also significantly increased. After 30 minutes of reperfusion, PGE2 levels were also elevated (p less than 0.05). Tissue edema measured by microgravimetry was also increased after 30 minutes of reperfusion in both gray and white matter. By 4 hours of reperfusion, rabbits regained near-normal hindlimb motor function while PGE2, 6-keto-PGF1 alpha, TxB2, and tissue water content were back to normal. However, by 18 hours of reperfusion, when hindlimb function was deteriorating, TxB2 levels were elevated again, and edema in gray and white matter was increased as was the number of necrotic neurons observed by light microscopy. These results suggest that the secondary deterioration of motor neurologic function was due to the excess formation of TxA2 primarily in the late reperfusion phase. However, further studies are necessary to elucidate the relation of TxA2 with ischemic neural injury.

Peripheral vascular pathophysiology of Plasmodium berghei infection: a comparative study in the cheek pouch and brain of the golden hamster.

Four- to six-week-old hamsters were infected with 1.5 X 10(7) Plasmodium berghei-parasitized hamster red blood cells by intraperitoneal injection. Cheek pouch circulation was observed microscopically in the anesthetized animal; the brain and contralateral pouch were collected for histopathologic examination on days 3-12 post-challenge. Cheek pouch vascular lesions, observed in vivo, appear to involve three phenomena; early (beginning 3-4 days) adhesion of pigment-laden mononuclear cells to endothelium within venous vessels and loss of function of the small capillaries supplying the skeletal muscle fibers and, later (6-9 days), the apparent attraction of erythrocytes to venular and venous endothelium and to adherent monocytes. The aggregation of formed elements on endothelial walls leads to progressive occlusion of venules and small veins and contributes to the observed disruption of flow through capillary networks. Histopathology of the brain and pouch shows vascular changes similar to those seen in vivo; in addition, multifocal hemorrhages are seen commonly in the brain and occasionally in the pouch on postmortem. In severe disease, evidence of cerebral edema is seen in the brain. The data suggest that failure of capillary flow and disruption of venous outflow tracts by cell aggregates are central to vascular failure in both the cheek pouch and brain of the P. berghei infected hamster. This hamster model of human cerebral malaria allows the in vivo observation, still and video photomicrography, and manipulation of the peripheral vascular pathogenesis of a disease process similar to that seen in humans.

An electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: maturation events.

The maturation process of dengue-2 virus in C6/36 mosquito cells was studied by electron microscopy at 12, 16, 24, 48, and 78 hours postinoculation (p.i.) and by immunoelectron microscopy at 48 and 78 hours p.i. Maturing virions appeared within cytoplasmic vacuoles and on the surface of infected cells from 24 hours p.i. onward in close topographical relationship to the dense particles that occurred concurrently in the cytoplasm. The dense particles measured 25 to 35 nm in diameter; the mature virions measured 50 to 55 nm in diameter, with a dense core measuring 30 to 35 nm in diameter covered by a 10 nm-thick membrane envelope. The morphological observations indicated that the dense particles were dengue nucleocapsids assembled in the cytoplasm and that they apparently budded into the vacuolar lumens and the extracellular space at the vacuolar and plasma membranes, acquiring membrane envelopes and becoming mature virions in the process. The virions that budded into the vacuolar lumens were released extracellularly by exocytosis. In the samples tested with dengue-2 polyclonal antibodies, intense immunostaining occurred at the sites of virus budding on the cell surface; host cell membrane and cytoplasm adjacent to the budding virions stained less intensely. In the samples tested with a dengue-2 monoclonal antibody specific for the envelope glycoprotein, budding virions stained rather exclusively, with no staining occurring in adjacent host membrane or cytoplasm.

Maturation process of Japanese encephalitis virus in cultured mosquito cells in vitro and mouse brain cells in vivo.

The maturation process of Japanese encephalitis (JE) virus in C6/36 cells in vitro and in mouse brain cells in vivo was studied by electron microscopy. In the C6/36 cell infection, 500 to 2250 virions per cell were released into the medium during the period of study; yet, no virus budding process was observed at the host cell membranes. JE virions at various maturation stages appeared within the cisternae of rough endoplasmic reticulum (RER) of infected cells at 24 hours p.i.; and, although C6/36 cells did not show a well-developed Golgi apparatus, the virions appeared to be carried to the cell surface within host-cell secretory vesicles for extracellular release as early as 24 hours p.i. The occurrence of a secretory-type intracellular transport of maturing JE virus particles was well recognizable in brain cells of infected mice, in which JE virus particles were found almost exclusively in the cisternae of RER, in the Golgi apparatus, and in various vesicles, including coated vesicles, in the vicinity of the Golgi apparatus. Our previous study of dengue-2 virus morphogenesis and our present study of JE virus morphogenesis differed substantially at various stages of maturation. Possible mechanisms which explain these differences were discussed.