Contributions of Nonhuman Primates to Research on Aging.

Aging is the biological process of declining physiologic function associated with increasing mortality rate during advancing age. Humans and higher nonhuman primates exhibit unusually longer average life spans as compared with mammals of similar body mass. Furthermore, the population of humans worldwide is growing older as a result of improvements in public health, social services, and health care systems. Comparative studies among a wide range of organisms that include nonhuman primates contribute greatly to our understanding about the basic mechanisms of aging. Based on their genetic and physiologic relatedness to humans, nonhuman primates are especially important for better understanding processes of aging unique to primates, as well as for testing intervention strategies to improve healthy aging and to treat diseases and disabilities in older people. Rhesus and cynomolgus macaques are the predominant monkeys used in studies on aging, but research with lower nonhuman primate species is increasing. One of the priority topics of research about aging in nonhuman primates involves neurologic changes associated with cognitive decline and neurodegenerative diseases. Additional areas of research include osteoporosis, reproductive decline, caloric restriction, and their mimetics, as well as immune senescence and chronic inflammation that affect vaccine efficacy and resistance to infections and cancer. The purpose of this review is to highlight the findings from nonhuman primate research that contribute to our understanding about aging and health span in humans.

Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA.

INTRODUCTION: Quantification of plasma viral load (PVL) is used to monitor disease progression in SIV-infected macaques. This study was aimed at optimizing of performance characteristics of the quantitative PCR (qPCR) PVL assay. METHODS: The PVL quantification procedure was optimized by inclusion of an exogenous control hepatitis C virus armored RNA (aRNA), a plasma concentration step, extended digestion with proteinase K, and a second RNA elution step. Efficiency of viral RNA (vRNA) extraction was compared using several commercial vRNA extraction kits. Various parameters of qPCR targeting the gag region of SIVmac239, SIVsmE660, and the LTR region of SIVagmSAB were also optimized. RESULTS: Modifications of the SIV PVL qPCR procedure increased vRNA recovery, reduced inhibition and improved analytical sensitivity. The PVL values determined by this SIV PVL qPCR correlated with quantification results of SIV RNA in the same samples using the 'industry standard' method of branched-DNA (bDNA) signal amplification. CONCLUSIONS: Quantification of SIV genomic RNA in plasma of rhesus macaques using this optimized SIV PVL qPCR is equivalent to the bDNA signal amplification method, less costly and more versatile. Use of heterologous aRNA as an internal control is useful for optimizing performance characteristics of PVL qPCRs.

Disseminated microsporidiosis due to Encephalitozoon hellem in an Egyptian fruit bat (Rousettus aegyptiacus).

Disseminated microsporidiosis was diagnosed in an adult female Egyptian fruit bat that died unexpectedly in a zoo. Gross findings, which were minimal, included poor body condition, bilateral renomegaly, and mottling of the liver. Histopathological lesions, which were particularly pronounced in the urogenital tract and liver, consisted primarily of inflammation associated with intracytoplasmic microsporidian spores. Polymerase chain reaction -based methods were used to establish the identity of the microsporidian as Encephalitozoon hellem. E. hellem is an emerging cause of human and avian disease, manifested mainly as opportunistic infection in immunosuppressed patients. This report describes the first documented case of E. hellem in a non-human mammalian species.

Encephalitozoon hellem infection in aviary passerine and psittacine birds in Spain.

A European goldfinch (Carduelis carduelis), a canary (Serinus canaria), and a lovebird (Agapornis roseicollis) captive-bred at three different private aviaries in Spain were submitted for necropsy with a history of weakness and ruffled feathers, weight loss associated with glossitis, and respiratory disease, respectively. Microscopically, enterocytes in the jejunum and ileum contained colonies of gram- and Stamp-positive, oval to elliptical microorganisms within parasitophorous vacuoles in the apical cytoplasm. Nested PCR using MSP primers that target microsporidian RNA genes produced amplicons of expected size for Encephalitozoon species, and analysis of forward and reverse DNA sequences confirmed the presence of Encephalitozoon hellem in all cases. The main cause of death of all three birds consisted of concurrent infections. However, intestinal encephalitozoonosis may have contributed to exacerbated catabolism. Encephalitozoonosis (or microsporidiosis) has been rarely described in passerine birds.

Microsporidia - Emergent Pathogens in the Global Food Chain.

Intensification of food production has the potential to drive increased disease prevalence in food plants and animals. Microsporidia are diversely distributed, opportunistic, and density-dependent parasites infecting hosts from almost all known animal taxa. They are frequent in highly managed aquatic and terrestrial hosts, many of which are vulnerable to epizootics, and all of which are crucial for the stability of the animal-human food chain. Mass rearing and changes in global climate may exacerbate disease and more efficient transmission of parasites in stressed or immune-deficient hosts. Further, human microsporidiosis appears to be adventitious and primarily associated with an increasing community of immune-deficient individuals. Taken together, strong evidence exists for an increasing prevalence of microsporidiosis in animals and humans, and for sharing of pathogens across hosts and biomes.

Macrophage cell line B6MP102 resembles peritoneal macrophages in tumor cell recognition and killing.

In this paper we describe the bone marrow-derived macrophage cell line B6MP102. We describe growth characteristics, responsiveness to biological response modifiers known to activate macrophages (MPs), and the ability of B6MP102--comparable to that of peritoneal MPs--to kill and discriminate tumor cells. We demonstrate that B6MP102 is easily maintained in culture in the presence of 15% L-M cell-conditioned media. We have found that the B6MP102 cell line is similar to in vivo-derived peritoneal MPs. These cells are responsive to biological response modifiers but are not spontaneously cytotoxic. They produce O2- in quantities comparable to that produced by peritoneal MPs, and they exhibit target ranges for direct killing similar to those of activated peritoneal MPs. In addition, B6MP102 is similar to MPs in mediating cytostasis. These latter two points are especially significant, suggesting that the mechanisms that MPs may use to distinguish and kill tumor cells are retained in the B6MP102 cell line. This makes the cell line a useful tool for studying MP-tumor cell interactions.

Characterization of two highly phosphorylated cytoskeleton-associated proteins, pp58 and pp60, in tumoricidal murine peritoneal macrophages and their comparison with vimentin.

Two TX-insoluble cytoskeleton-associated proteins, pp58 and pp60, become highly phosphorylated in tumoricidal murine peritoneal macrophages. Results suggest that pp58 (pI 5.00) is phosphovimentin because it is highly insoluble in TX, shares the same mol. wt as vimentin, has a more acidic isoelectric point than vimentin, is phosphorylated primarily at serine, and generates the same V-8 protease peptide map as vimentin. pp60 generates at slightly different peptide map than pp58 and has a slightly less acidic isoelectric point (pI 5.02) than pp58 (pI 5.00), but is similar to pp58 by being highly insoluble in TX and being phosphorylated primarily at serine residues. Pulse-chase experiments demonstrate that pp58 is not a precursor to or breakdown product of pp60, or vice versa because they show similar rates of [32P]-phosphate incorporation and turnover.

Microsporidiosis in mammals.

Microsporidia are small, single-celled, obligately intracellular parasites that have caused significant agricultural losses and interference with biomedical research. Interest in the microsporidia is growing, as these organisms are recognized as agents of opportunistic infections in persons with AIDS and in organ transplant recipients. Microsporidiosis is also being recognized in children and travelers, and furthermore, concern exists about the potential of zoonotic and waterborne transmission of microsporidia to humans. This article reviews the basic biology and epidemiology of microsporidiosis in mammals.

Biology of microsporidian species infecting mammals.

Microsporidia (phylum Microspora) are obligate intracellular protozoan parasites that infect a wide range of vertebrate and invertebrate hosts. Over 1000 species have been classified into approximately 100 genera, and at least 13 species have been reported to infect mammals. Phylogenetically, the microsporidia are early eukaryotes because they have a true nucleus, possess prokaryote-like ribosomes, and lack mitochondria. The species that infect mammals are relatively small, measuring 2.0-7.0 microns long and 1.5-5.0 microns wide. The mature organism is the spore, which is enclosed by a chitinous coat, making it relatively resistant to the environment. Infections often occur by fecal-oral or urinary-oral transmission, although vertical transmission is quite common in the carnivores. Host cells become infected through a process of germination in which the spore propels its contents through the everting and unwinding polar filament into the host cell. The polar filament is unique to the microsporidia. With a few exceptions, microsporidiosis is typically chronic and subclinical in immunologically competent hosts. Young carnivores infected with microsporidia, however, develop severe and sometimes lethal renal disease, and immunodeficient laboratory animals (e.g. athymic and SCID mice) develop ascites and die from microsporidiosis. This review describes the morphology, life cycle, taxonomy, and host-parasite relationships of the species of microsporidia that infect mammals.

Pulmonary infection with microsporidia after allogeneic bone marrow transplantation.

Microsporidia are obligate, intracellular protozoal parasites that can be pathogenic in immunocompromised individuals. The majority of cases of microsporidiosis have been documented in patients with HIV, and only a few case reports exist of infection in solid organ transplant patients. We report the first case of pulmonary microsporidial infection in an allogeneic bone marrow transplant recipient in the US. The patient was a recipient of a T-cell-depleted graft who succumbed to complications from respiratory failure 63 days post transplant. The diagnosis was made post mortem by electron microscopy and confirmed with PCR. Although rare, microsporidial infection should be considered in the differential diagnosis of unexplained pulmonary infection in bone marrow transplant patients.

Renal Encephalitozoon (Septata) intestinalis infection in a patient with AIDS. Post-mortem identification by means of transmission electron microscopy and PCR.

We describe the occurrence of renal Encephalitozoon (Septata) intestinalis infection in a 35-year-old AIDS patient who died with disseminated tuberculosis. The patient did not complain of specific symptoms involving the kidney or lower urinary tract during life, but at autopsy, light microscopic examination of the kidney revealed numerous small round or oval bodies in the tubules and tubular cell cytoplasm that were interpreted as intracellular protozoa. Transmission electron microscopy of tissue retrieved from paraffin-embedded samples identified these organisms as microsporidia belonging to the Encephalitozoonidae family, but did not allow definitive identification of the species of infecting parasite. This was made possible only by means of Southern blot hybridization after the polymerase chain reaction, which recognized the micro-organism as E. intestinalis.

Speciation of human microsporidia by polymerase chain reaction single-strand conformation polymorphism.

We describe the application of single-strand conformation polymorphism (SSCP) analysis to the speciation of human microsporidia after polymerase chain reaction (PCR) amplification with the panmicrosporidian primers PMP1 and PMP2. We compared the DNA extracted and amplified from different genotypes or isolates of Enterocytozoon bieneusi, Encephalitozoon cuniculi, E. hellem, and E. intestinalis plus an isolate of Vittaforma corneae. The PCR-SSCP, when performed at 20 degrees C, generated 2 bands in distinctive, reproducible patterns in polyacrylamide gels for each species of microsporidia tested, regardless of genotype or isolate. We found PCR-SSCP to be an easy and reproducible method for speciation of human microsporidia when the primer pair PMP1 and PMP2 is used.

Microsporidial keratoconjunctivitis caused by Septata intestinalis in a patient with acquired immunodeficiency syndrome.

PURPOSE: To examine and treat a patient with acquired immunodeficiency syndrome (AIDS) who had mildly hyperemic conjunctiva and epithelial keratopathy in both eyes. METHODS: The patient underwent conjunctival biopsy. The specimen was examined by transmission electron microscopy. RESULTS: Septata intestinalis was demonstrated to be the cause of keratoconjunctivitis in the patient. The keratoconjunctivitis resolved after three weeks of therapy with topical fumagillin. No organisms were seen on repeat conjunctival biopsy. CONCLUSIONS: Microsporidial keratoconjunctivitis in patients with AIDS can be caused by S. intestinalis. This condition appears to respond to topical fumagillin.

Epidemiology of microsporidiosis: sources and modes of transmission.

Microsporidia are single-celled, obligate intracellular parasites that were recently reclassified from protozoa to fungi. Microsporidia are considered a cause of emerging and opportunistic infections in humans, and species infecting humans also infect a wide range of animals, raising the concern for zoonotic transmission. Persistent or self-limiting diarrhea are the most common symptoms associated with microsporidiosis in immune-deficient or immune-competent individuals, respectively. Microsporidian spores appear to be relatively resistant under environmental conditions, and species of microsporidia infecting humans and animals have been identified in water sources, raising concern about water-borne transmission. Sensitive and specific immunomagnetic bead separation and PCR-based methods are being developed and applied for detecting microsporidia in infected hosts and water sources for generating more reliable prevalence data. The most effective drugs for treating microsporidiosis in humans currently include albendazole, which is effective against the Encephalitozoon species but not against Enterocytozoon bieneusi, and fumagillin, which has broader anti-microsporidia activity but is toxic in mammals, suggesting a need to identify better drugs. Strategies to capture and disinfect microsporidia in water are being developed and include filtration, coagulation, chlorination, gamma-irradiation, and ozonation.

Intestinal microsporidiosis in African skink Mabuya perrotetii.

Intestinal microsporidiosis was documented by detecting abundant slightly curved spores (2.9 x 1.2 microns) in the faeces of five of twelve skinks Mabuya perrotetii Duméril et Bibron, 1839 that originated from Ghana. Clinically, the microsporidiosis was characterized by decreased appetite, diarrhea, and weight loss. Histopathological changes consisted of villous atrophy, blunting of mucosa and flattening of individual epithelial cells in the large intestine. The ultrastructure of microsporidian spores was consistent with an Encephalitozoon species. The PCR-RFLP assay and the heteroduplex mobility shift analyses were used to verify that the skink microsporidian is a species of the genus Encephalitozoon Levaditi, Nicolau et Schoen, 1923 and indicate that this microsporidian is not E. hellem, E. intestinalis or a strain of E. cuniculi. The microsporidia in African skink represent an Encephalitozoon species morphologically identical to Encephalitozoon lacertae Canning, 1981.

Experimental microsporidiosis in immunocompetent and immunodeficient mice and monkeys.

Microsporidia cause opportunistic infections in AIDS patients and commonly infect laboratory animals, as well. Euthymic C57B1/6 mice experimentally infected with intraperitoneal injections of 1 x 10(6) Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923, Encephalitozoon hellem Didier et al., 1991, or Nosema corneum Shadduck et al., 1990 displayed no clinical signs of disease. Athymic mice, however, developed ascites and died 8-16 days after inoculation with N. corneum, 21-25 days after inoculation with E. cuniculi, and 34-37 days after inoculation with E. hellem. All athymic mice displayed hepatomegaly, dilated intestine and accumulation of ascites fluid. Granulomatous lesions are primarily located in the liver, lung, pancreas, spleen, and on serosal surfaces of abdominal organs. The murine microsporidiosis model also was used to examine immune response that inhibit microsporidia growth in vitro. Recombinant murine interferon-gamma (mIFN-gamma, 100 mu/ml) alone or in combination with lipopolysaccharide (LPS; 10 ng/ml) could activate thioglycollate-induced peritoneal murine macrophages to destroy E. cuniculi. The production of the nitrogen intermediate, NO2-, correlated with parasite destruction. Inhibition of NO2- generation by addition of the L-arginine analogue, NG-monomethyl L-arginine (NMMA), inhibited microsporidia killing, as well. Since microsporidiosis is becoming an important opportunistic infection in AIDS patients, a microsporidiosis model is being developed using SIV/DeltaB670-infected rhesus macaque monkeys (Macaca mulatta). SIV-infected immunocompetent monkeys given E. cuniculi or E. hellem per os developed specific antibodies, and microsporidia could be detected sporadically by calcofluor or antibody fluorescence staining of stool and urine sediment smears. As immunodeficiency progressed, monkeys developed diarrhoea, cachexia, and anorexia, and organisms were detected in urine and stool with greater frequency. Immunodeficient SIV-infected monkeys died approximately 27 days after receiving E. hellem by intravenous inoculation, and approximately 110 days after receiving E. hellem per os. Lesions typical for SIV-infection were observed in both groups of monkeys and microsporidia were detected in kidney and liver of the intravenously-injected monkeys. The murine microsporidiosis model provides an efficient means for studying protective immune responses to microsporidiosis, and may prove useful for screening immunological and chemotherapeutic agents. The pathogenesis of Encephalitozoon microsporidiosis in SIV-infected monkeys appears to parallel encephalitozoonosis in AIDS patients, suggesting that simian microsporidiosis may provide a useful model for evaluating diagnostic methods and therapeutic strategies during various stages of progressing immunodeficiency.

Screening of compounds for antimicrosporidial activity in vitro.

Relatively few effective compounds are available for treating microsporidiosis in humans. In this study, several compounds were assayed for activity against Encephalitozoon intestinalis (Cali, Kotler et Orenstein, 1993) and Vittaforma corneae Shadduck, Meccoli, Davis et Font, 1990 in vitro. Of the benzimidazoles tested, albendazole was most effective and the MIC50 values were 8.0 ng/ml and 55.0 ng/ml for E. intestinalis and V. corneae, respectively. Fumagillin and its analogue, TNP-470 were nearly equally effective against both E. intestinalis and V. corneae. The MIC50 values of fumagillin were 0.52 ng/ml and 0.81 ng/ml, and the MIC50 values of TNP-470 were 0.35 ng/ml and 0.38 ng/ml for E. intestinalis and V. corneae, respectively. In addition, 12 of 44 purines and pteridines with putative tubulin binding activity that were synthesized at Southern Research Institute (SRI), inhibited microsporidial replication by more than 50% at concentrations that were not toxic to the host cells. Several chitin synthesis/assembly inhibitors inhibited growth of the microsporidia in vitro but were toxic for the host cells making it difficult to interpret the results. One exception was lufenuron, which caused no significant toxicity to the host cells and expressed approximate MIC50 values of 2.95 micrograms/ml and 6.3 micrograms/ml against E. intestinalis and V. corneae, respectively. These results warrant further studies on albendazole, fumagillin, TNP-470, lufenuron, and the selected SRI purines and pteridines for developing therapeutic strategies for microsporidiosis.