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The facial muscles have significant roles for vocalization, feeding, and facial expression in both human and non-human primates. Of these, the anatomy of the incisivus labii superioris (ILS) and incisivus labii inferioris (ILI), which are considered as the accessory bundle of the orbicularis oris (OO) in humans, has rarely been documented in the literature. Our current understanding of the function of the ILS and ILI is that they probably retract the upper and lower lips. Also, there is no account of these muscles in non-human primates in the current literature. The aim of this study was to reveal the ILS and ILI in non-human primates. Five Macaca fascicularis, one Macaca fuscata, one Macaca fuscata yakui, and one Pan troglodytes were dissected. Seven formalin-fixed cadavers and one fresh cadaver were included. Both the ILS and ILI were observed in all specimens. The ILS originated from the incisive fossa of the maxilla and inserted into the OO. The mentalis (MT) and ILI arose from the incisive fossa of the mandible and inserted into the OO and the skin of the chin area. The MT and ILI in the P. troglodytes examined were thicker than in the other three non-human species, and the ILS and ILI in the three macaques were similar in shape to those of humans. The difference of these muscles may result in different functions of the lip such as during vocalization, feeding, and facial expression., (© 2020 American Association for Anatomy.)

Isoflurane anesthesia alters the blood levels of several neuroendocrine hormones associated with normal metabolism and physiology and increases stress, but the effect of brief CO2 anesthesia on these parameters is unknown. In this study, we examined the effects of isoflurane (4%) compared with brief CO2 (70% CO2, 30% air) anesthesia on circadian rhythms of plasma measures of physiology and metabolism. Adult male Sprague-Dawley rats (Crl:SD; n = 6 per group) were maintained on a 12:12-h light:dark (300 lx; lights on, 0600) photoperiod. After 1 wk of acclimation, a series of 6 low-volume blood draws were collected by cardiocentesis under anesthesia using isoflurane (10 min or less) compared with CO2 (1 min or less) at a single circadian time point every 4 d (0400, 0800, 1200, 1600, 2000, or 2400) over 3 wk to assess arterial blood glucose, lactic acid, and potassium and plasma melatonin, leptin, insulin, total fatty acids, and corticosterone concentrations. Results revealed that plasma levels (mean ± SEM) of melatonin were low (11 ± 1 pg/mL) during the light phase in both groups but were significantly lower during the dark phase in the isoflurane group (48 ± 6 pg/mL) compared with the CO2 group (162 ± 18 pg/mL). In addition, prominent circadian rhythms of arterial plasma levels of corticosterone, glucose, total fatty acids, lactic acid, and potassium were altered in the isoflurane group compared with the CO2 group. These findings demonstrate that the normal circadian rhythms of endocrine physiology and metabolism observed during brief CO2 anesthesia in rats are markedly disrupted by isoflurane anesthesia.

Environmental enrichment (EE) gives laboratory animals opportunities to engage in species-specific behaviors. However, the effects of EE devices on normal physiology and scientific outcomes must be evaluated. We hypothesized that the spectral transmittance (color) of light to which rats are exposed when inside colored enrichment devices (CED) affects the circadian rhythms of various plasma markers. Pair-housed male Crl:SD rats were maintained in ventilated racks under a 12:12-h light:dark environment (265.0 lx; lights on, 0600); room lighting intensity and schedule remained constant throughout the study. Treatment groups of 6 subjects were exposed for 25 d to a colored enrichment tunnel: amber, red, clear, or opaque. We measured the proportion of time rats spent inside their CED. Blood was collected at 0400, 0800, 1200, 1600, 2000, and 2400 and analyzed for plasma melatonin, total fatty acids, and corticosterone. Rats spent more time in amber, red, and opaque CED than in clear tunnels. All tubes were used significantly less after blood draws had started, except for the clear tunnel, which showed no change in use from before blood sampling began. Normal peak nighttime melatonin concentrations showed significant disruption in the opaque CED group. Food and water intakes and body weight change in rats with red-tinted CED and total fatty acid concentrations in the opaque CED group differed from those in other groups. These results demonstrate that the color of CED altered normal circadian rhythms of plasma measures of metabolism and physiology in rats and therefore might influence the outcomes of scientific investigations.

The suprachiasmatic nucleus is synchronized by the light:dark cycle and is the master biologic clock that serves as a pacemaker to regulate circadian rhythms. We explored the hypothesis that spectral transmittance (tint) of light through caging alters circadian rhythms of endocrine and metabolic plasma constituents in nonpigmented Sprague-Dawley rats. Rats (Crl:SD; n = 12 per group) were housed in a 12:12-h light:dark environment (300 lx; 123.0 μ W/cm(2); lights on, 0600) in either clear-, amber-, blue-, or red-tinted rodent cages. Blood was collected at 0400, 0800, 1200, 1600, 2000, and 2400 and measured for melatonin, total fatty acids, pH, glucose, lactic acid, corticosterone, insulin, and leptin. As expected, plasma melatonin levels were low during the light phase but higher during the dark phase in all groups; however, when compared with the clear-cage group, rats in amber-, blue-, and red-tinted cages had 29%, 74%, and 48%, respectively, greater total daily melatonin levels due to an increased duration and, in some cases, amplitude of the nocturnal melatonin signal. No differences were found in dietary and water intake, body growth rates, total fatty acids, pH, or glucose among groups. Disruptions in circadian rhythms, manifesting as alterations in phase timing, amplitude, or duration, occurred in the melatonin, lactic acid, corticosterone, insulin, and leptin levels of rats in tinted compared with clear cages. Therefore, the use of variously tinted animal cages significantly alters circadian rhythms in plasma measures of metabolism and physiology in laboratory rats, thus potentially altering the outcomes of scientific investigations.

Self-injurious behavior (SIB) is a spontaneous behavior that threatens the health and wellbeing of multiple species. In humans, the opioid antagonist naltrexone hydrochloride has been used successfully to modulate the endogenous opioid system and reduce the occurrence of SIB. This study is the first to assess the efficacy of extended-release naltrexone in the pharmacologic treatment of SIB in rhesus macaques (Macaca mulatta). In an acute pharmacokinetic study of 4 macaques, we determined the mean naltrexone plasma concentration was maintained above the therapeutic level (2 ng/mL) after administration of a single dose (20 mg/kg) of 28-d extended-release naltrexone throughout the release period. For a subsequent treatment study, we selected 8 singly housed macaques known to engage in SIB. The study comprised a 4-wk baseline phase; an 8-wk treatment phase, during which each macaque received 2 doses of extended-release naltrexone 28 d apart; and a 4-wk posttreatment phase. Plasma samples were collected and analyzed weekly for naltrexone concentrations throughout the treatment and posttreatment phases. In addition, total of 6 h of video was analyzed per animal per phase of the study. Compared with baseline phases, both the frequency and the percentage of time spent displaying SIB decreased during the treatment phase, and the percentage of time remained decreased during the posttreatment phase. In contrast, extended-release naltrexone did not alter the expression of other abnormal, anxiety-related, or agonistic behaviors nor were levels of inactivity affected. The present study supports the use of naltrexone in the treatment of SIB in rhesus macaques.

Background: Nasopharyngeal colonization by Streptococcus pneumoniae precedes pneumococcal disease. Elucidation of procedures to prevent or eradicate nasopharyngeal carriage in a model akin to the human would help to diminish the incidence of both pneumonia and invasive pneumococcal disease., Methods: We conducted a survey of the nasopharynx of infant rhesus macaques from our breeding colony, in search of natural carriers of S. pneumoniae. We also attempted experimental induction of colonization, by nasopharyngeal instillation of a human S. pneumoniae strain (19F)., Results: None of 158 colony animals surveyed carried S. pneumoniae in the nasopharynx. Colonization was induced in eight of eight infant rhesus by nasopharyngeal instillation and lasted 2weeks in 100% of the animals and 7weeks in more than 60%., Conclusion: Rhesus macaques are probably not natural carriers of S. pneumoniae. The high rate and duration of colonization obtained in our experiments indicates that the rhesus macaque will serve as a human-like carriage model., (© 2011 John Wiley & Sons A/S.)

Bronchoalveolar lavage (BAL) by means of bronchoscopy is a diagnostic tool frequently used for clinical and research purposes in nonhuman primates. Although many institutions use this procedure, the technique is not standardized. One technical aspect that can vary is the method by which fluid is recovered. The purpose of this study was to evaluate differences between 2 different BAL aspiration techniques. Bronchoscopy and BAL fluid collection were performed on 20 rhesus macaques (Macaca mulatta). Data collected for comparison included heart rate, oxygen saturation levels, rectal temperature, volume of fluid collected, total cell count, cell viability, differential cell count, and flow cytometry. Results showed no significant differences in the heart rate, oxygen saturation, or body temperature between the 2 groups. Likewise, differential cell counts and cell viability studies of the retrieved fluid did not differ between methods. Compared with the conventional technique, the modified aspiration technique led to an 8.3% increase in overall fluid yield and a higher concentration of cells recovered. These differences are statistically significant and likely will be clinically relevant in the context of diagnosis.

As part of a study addressing chronic alcohol consumption and simian immunodeficiency virus, 31 rhesus macaques (Macaca mulatta) were implanted with gastric catheters used to deliver alcohol or isocaloric sucrose (control). Once implanted, the animals wore jackets and were housed in specialized cages modified with swivels and tethers. During the course of the study, 3 animals developed clinical signs indicating possible instability of the implanted gastric catheter. All 3 animals were found to have a string foreign body wrapped around the distal end of the catheter, with 2 of the catheters perforating the intestinal wall. Gastroscopy was used to screen remaining animals to determine catheter position and the presence of a foreign body attached to the end of the catheter. Results of the screening revealed that of the 28 remaining animals, 9 had malpositioned catheters; string foreign bodies were associated with 3 of the 9 malpositioned catheters. We initially hypothesized that the peristaltic motion of the stomach, combined with the attachment of string, which was probably ingested by subjects after manipulating their jackets, led to eventual catheter displacement. We later concluded that the string may have played a secondary role but was not the primary cause of catheter instability, because several malpositioned catheters had no string attached at the time of diagnosis. Subsequent modifications were instituted, including modifying the surgical technique, altering the type of gastric catheter used, and increasing environmental enrichment for animals with known tendency to manipulate their jackets.

Leptin is a polypeptide hormone produced by adipose and other endocrine tissues. Although it has been linked to receptor-mediated pathways that directly influence human conceptus development, mechanisms that regulate the leptin receptor in pregnancy-specific tissues remain unclear. Therefore, we assessed leptin-receptor ontogeny and regulation in the baboon (Papio sp.), a primate model for human pregnancy. Placentae, decidua, and amniochorion were collected from baboons in early (Days 54-63, n = 4), mid (Days 98-103, n = 4), and late (Days 159-165, n = 4) gestation. Regulation by estrogen was assessed by elimination of androgen precursors via removal of the fetus (fetectomy) at midgestation and collection of tissues in late gestation (n = 4; term, approximately 184 days). Maternal serum was sampled with advancing gestation, and the abundance of soluble leptin receptor (solLepR), a potential mediator of gestational hyperleptinemia, was determined. Two placental leptin-receptor isoforms (130 and 150 kDa) increased (P < 0.04 and P < 0.02, respectively) in abundance with advancing gestation. Similarly, the 130-kDa isoform increased approximately fourfold (P < 0.0025) in decidua and approximately 10-fold (P < 0.015) in amniochorion between early and late gestation. Following fetectomy, maternal serum estradiol levels declined approximately 85% (P < 0.03), and the 150-kDa placental leptin-receptor isoform was reduced by more than half (P < 0.002). Maternal serum solLepR concentrations were correlated with gestational age (r = 0.52, P < 0.01) and were unaffected by fetectomy. The presence of leptin-receptor isoforms in pregnancy-specific tissues further denoted leptin's potential to directly influence conceptus development, whereas the 130-kDa solLepR identified in maternal serum suggested a means to facilitate the hyperleptinemia typical of primate pregnancy. Although estrogen did not appear to be the principal regulator of solLepR, it and other factors linked to advancing gestation may be implicated in the regulation of leptin-receptor synthesis.

Serological diagnosis of West Nile virus (WNV) infection is complicated by extensive antigenic cross-reactivity with other closely related flaviviruses, such as St. Louis encephalitis virus. Here we describe a recombinant, bacterially expressed antigen equivalent to structural domain III of the WNV envelope protein that has allowed clear discrimination of antibody responses to WNV from those against other related flaviviruses in indirect enzyme-linked immunosorbent assays using standardized control antisera and field-collected samples.

During the summer of 2002, an epidemic of West Nile meningoencephalitis occurred in southern Louisiana. Following the outbreak, blood samples were collected from 1,692 captive rhesus monkeys (Macaca mulatta), pigtail macaques (M. nemestrina), and baboons (Papio spp.) that were permanently housed outdoors at a nonhuman primate breeding facility in St. Tammany Parish, Louisiana. The serum samples were examined for antibodies to West Nile virus (WNV). Overall, 36% of the captive nonhuman primates had WNV antibodies; comparison of these samples with banked serum samples from previous blood collections indicated that the animals were infected subclinically from February to August 2002. WNV activity was demonstrated in surveillance at the nonhuman primate-breeding colony and in the neighboring community during this same period. The high infection rate in this captive nonhuman primate population illustrates the intensity of WNV transmission that can occur silently in nature among other susceptible vertebrates during epidemic periods.

Background: Alcohol abuse and infection with HIV individually compromise immune function, but the consequence of both conditions together is poorly understood owing to the difficulties of performing appropriate studies in human subjects. Simian immunodeficiency virus (SIV) infection of rhesus macaques is considered to closely model HIV disease in that the virus infects CD4+ cells and this infection leads to a similar AIDS state. This study was initiated to study the combined effects of chronic binge alcohol consumption on the primary stage of SIV infection., Methods: Rhesus macaques were administered alcohol or isocaloric sucrose via a permanently indwelling intragastric catheter 4 consecutive days per week for the duration of the study. Doses were individualized to achieve plasma alcohol concentrations of 50-60 mM over a 5-hr period. After 3 months, animals were inoculated intravenously with 10,000 times the ID(50) (50% infective dose) of SIV(DeltaB670) at the conclusion of an alcohol session and followed for 2 months postinoculation., Results: At 1 week, plasma SIV RNA was greater than 60-fold higher in alcohol-consuming animals compared with sucrose controls. Likewise, alcohol consumption enhanced the SIV-induced increase in cell cycling T lymphocytes (i.e., cells expressing Ki67 protein) in blood. These differences between alcohol- and sucrose-treated animals were not sustained during the observation period. Peak viral load occurred 2 weeks post-SIV inoculation at 7.6 +/- 4.2 and 5.2 +/- 3.1 x 106 copies/ml in alcohol- versus sucrose-consuming animals, respectively. Blood CD4+ lymphocyte numbers were decreased 1 and 2 months after SIV inoculation to a similar degree in both sucrose-control and alcohol-treated animals., Conclusions: The consequence of the early rise in viral load and increase in lymphocyte turnover seen with excess alcohol consumption is unknown. We hypothesize that alcohol intoxication may increase the susceptibility of the host to HIV/SIV infection. This possibility needs to be explored further.

Groups of rhesus monkeys (RM) were vaccinated and boosted with living Mycobacterium bovis Bacillus Calmette-Guerin (BCG) or BCG + low dose (LD) heat-killed Mycobacterium leprae (HKML) or high dose (HD) HKML or were unvaccinated. Animals vaccinated with BCG + LD and HD HKML were lepromin skin tested 2 weeks after boosting. All groups were lepromin tested 37 and 46 months after challenge with live M. leprae. Fernandez (72 h) and Mitsuda (28 day) responses were recorded. Ten of 10 rhesus monkeys in each of the two BCG + HKML-vaccinated groups significantly converted to strong positive Fernandez status within 2 weeks of boosting, compared to one of six positives in the unvaccinated unchallenged normal control group. Both BCG + HKML groups were significantly protected from clinical leprosy. Six of 10 in each of the two BCG + HKML groups significantly converted to Mitsuda positivity within 2 weeks of boosting compared to zero of six in the normal control group. The sizes of the Mitsuda responses were larger in the LD group than the HD HKML vaccinated/boosted group, suggesting suppression by vaccination with higher doses of HKML in combination with BCG. Fernandez responses were negative in normal RM as well as in the unvaccinated, ML-challenged group and the BCG-vaccinated, ML-challenged group at 37 or 46 months after ML inoculation, although the BCG-vaccinated group was significantly protected from leprosy and the unvaccinated group was not. In contrast, at 37 months the Fernandez reaction was positive in the BCG plus LD and the BCG plus HD HKML-vaccinated groups, both of which were significantly protected from clinical leprosy. By 46 months, the Fernandez responses were below significance in all groups. Thus, Fernandez reactivity is not a reliable correlate to protection from experimental leprosy in RM. Mitsuda responses became strongly positive in all four ML-challenged groups by 37 months and remained strongly positive at 46 months after ML inoculation, suggesting that strong Mitsuda reactivity reflects responses to living ML. BCG or BCG + LD or HD HKML vaccination/boosting of RM produced significant clinical protection from leprosy and there was a good correlation between protection from LL forms of leprosy and positive Mitsuda skin test responses after challenge with live ML. Positive Mitsuda responses were generated in essentially all individuals after challenge with live ML, and this response was primed by prior vaccination/boosting with BCG + HKML as shown by conversion to positivity 2 weeks after boosting. The data show that resistance to clinical leprosy is reflected by Mitsuda responses in ML-exposed RM, similar to results from human studies, and confirm the suitability of RM as a model for leprosy vaccine studies.

Analysis of sequence variations among isolates of Pneumocystis carinii f. sp. macacae from 14 Indian rhesus monkeys (Macaca mulatta) at the internal transcribed spacer (ITS) regions of the nuclear rRNA gene was undertaken. Like those from P. carinii f. sp. hominis, the ITS sequences from various P. carinii f. sp. macacae isolates were not identical. Two major types of sequences were found. One type of sequence was shared by 13 isolates. These 13 sequences were homologous but not identical. Variations were found at 13 of the 180 positions in the ITS1 region and 28 of the 221 positions in the ITS2 region. These sequence variations were not random but exhibited definite patterns when the sequences were aligned. According to this sequence variation, ITS1 sequences were classified into three types and ITS2 sequences were classified into five types. The remaining specimen had ITS1 and ITS2 sequences substantially different from the others. Although some specimens had the same ITS1 or ITS2 sequence, all 14 samples exhibited a unique whole ITS sequence (ITS1 plus ITS2). The 5.8S rRNA gene sequences were also analyzed, and only two types of sequences that differ by only one base were found. Unlike P. carinii f. sp. hominis infections in humans, none of the monkey lung specimens examined in this study were found to be infected by more than one type of P. carinii f. sp. macacae. These results offer insights into the genetic differences between P. carinii organisms which infect distinct species.

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.

To support an infectious disease study, we developed a surgical procedure to collect serial thymic biopsies in rhesus monkeys (Macaca mulatta). In many instances in which thymic tissue is required from living animals, open surgical approaches (thoracotomies) are used, which result in greater postoperative pain and longer recovery periods than those associated with thoracoscopic procedures. Our intent was to develop a surgical procedure that allowed serial biopsy of the thymus with minimal surgical morbidity. We modified a previously published experimental method of thoracoscopic total thymectomy in the dog to collect thymic biopsies in M. mulatta. Of the 15 animals evaluated, 8 underwent two biopsy procedures separated by a 5- to 6-week interoperative interval. The other seven animals underwent a single biopsy procedure. Thymic tissue was collected successfully during all procedures, with an average surgical time of 15 min. No significant intra- or postoperative complications were noted, and the animals recoveries from the surgical procedures were uneventful. Minimally invasive thoracoscopic surgical techniques can be used successfully to collect thymic tissue from adult and juvenile rhesus monkeys with minimal surgical morbidity.

The baboon (Papio sp.) is an accepted nonhuman primate model for the study of the endocrinology of human pregnancy. To further characterize this model with regard to leptin function, messenger RNA transcripts for both long (Ob-RL) and short (Ob-RS) leptin receptor isoforms were identified in maternal tissues at various stages of gestation. Thus, placental villous, subcutaneous and omental adipose tissues were collected upon cesarean delivery at early (Days 60-62), mid (Days 98-102) and late (Days 159-164) pregnancy (term approximately 184 days). Additionally, amniochorion, decidua, and corpus luteum were collected in late gestation. Expression of Ob-RL and Ob-RS transcripts was determined in relation to constitutively expressed glyceraldehyde-3-phosphate dehydrogenase via reverse transcriptase-polymerase chain reaction, and transcripts were localized within specific placental cell types by in situ hybridization. Ob-RL and Ob-RS transcripts were present in amniochorion, decidua, and corpus luteum at term and appeared constitutively expressed throughout gestation in placenta and adipose tissues. Ob-RS was expressed in greater (P < 0.02) abundance than Ob-RL in all tissues. Within the placenta, receptor isoforms were localized predominantly to the syncytiotrophoblast. The expression of leptin receptor transcripts in maternal adipose tissues, as well as in the syncytiotrophoblast, amniochorion, decidua, and corpus luteum, suggests the potential for autocrine/paracrine roles for the polypeptide in the endocrinology of primate pregnancy. These are the first such observations in a nonhuman primate and support the use of the baboon as a model for the study of leptin in human pregnancy.

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.

Pulmonary infections are a significant cause of morbidity and mortality in patients with alcohol abuse and human immunodeficiency virus (HIV) infection, two immunocompromising conditions that frequently coexist. This study examined the separate and combined effects of in vivo lentiviral infection and in vitro alcohol exposure on alveolar macrophage (AM) production of tumor necrosis factor- alpha (TNF-alpha), a proinflammatory cytokine that is critical to normal pulmonary host defense. AMs, recovered by bronchoalveolar lavage (BAL) from uninfected and simian immunodeficiency virus (SIV)-infected rhesus macaques, at the asymptomatic and terminal stages of infection, were cultured in ethanol 2 h prior to stimulation with lipopolysaccharide (LPS). Median TNF-alpha concentrations were measured 15 h later. Spontaneous TNF-alpha production was similar in all groups examined. LPS increased TNF-alpha protein production similarly in SIV(-) (2, 381 +/- 359 pg/ml) and SIV(+) animals at the terminal stage of infection (2,019 +/- 507 pg/ml). In contrast, cells from SIV(+) asymptomatic animals had a depressed response (763 +/- 304 pg/ml). Ethanol (100 mM) suppressed the LPS-induced AM TNF-alpha response by approximately 50% in both SIV(-) and (+) animals. Ethanol-induced suppression of the TNF-alpha response occurred at a post-transcriptional level. These data suggest that ethanol-induced suppression of the pulmonary TNF-alpha response may further increase the susceptibility to and severity of secondary infectious complications in HIV-infected hosts.

Induced immunosuppression is required for a number of studies using rhesus monkeys (Macaca mulatta). This report describes the clinical outcome and safety of a dose-finding experiment that determined doses of cyclophosphamide and prednisone that could be used to induce a state of immunosuppression in rhesus monkeys. After determining the optimum dose of immunosuppressive agents, the protocol was then used on animals participating in infectious disease and gene therapy studies. Splenectomy was performed in some animals to increase the severity of immunosuppression. The onset, duration, and severity of lymphopenia and leukopenia were consistent in all animals. In most animals, physical examination findings and clinical serum chemistry profiles demonstrated only transient abnormalities. With proper clinical monitoring, combination treatment with cyclophosphamide and prednisone is an effective and safe method for inducing immunosuppression in rhesus monkeys.

Background: Human immunodeficiency virus (HIV) infection and alcohol abuse frequently coexist in the host and are known to suppress individually the host response to a variety of opportunistic infections., Methods: This study examined the effects of in vitro ethanol exposure on several functions of polymorphonuclear leukocytes (PMNs) that were obtained from uninfected and simian immunodeficiency virus (SIV)-infected rhesus macaques, at the asymptomatic and terminal stages of infection., Results: The PMNs obtained from rhesus macaques at both the asymptomatic and terminal stage of SIV disease had elevated phagocytic activity and increased CD11b expression compared with PMNs from uninfected animals. In vitro 100 mM ethanol suppressed phagocytosis and CD11b adhesion molecule expression by PMNs, regardless of the stage of SIV infection. Treatment of PMNs with granulocyte colony-stimulating factor (G-CSF) attenuated the inhibitory effect seen with prior ethanol exposure., Conclusions: These data demonstrate that the functional state of PMNs from uninfected as well as SIV-infected rhesus macaques is impaired by direct exposure to intoxicating concentrations of ethanol and that this effect can be attenuated by G-CSF. If alcohol intoxication similarly suppressed PMN function in vivo, it would further increase susceptibility of these hosts to secondary infections. Furthermore, G-CSF may be useful in overcoming the suppressive effects of ethanol on PMN function in such patients.

The histopathologic and immunohistochemical features of early and late neuroborreliosis of the peripheral nervous system were investigated in rhesus macaques infected with the JD1 strain of Borrelia burgdorferi. Infection was proven by culture or polymerase chain reaction analysis of skin biopsies and indirectly by Western blot analysis. Three months after infection, neuritis involving multiple nerves was the most consistent neurologic manifestation. Both macrophages and B lymphocytes but not T lymphocytes were present in the cellular infiltrates. Axonal structures surrounding infiltrates had changes consisting of demyelination and axonal phagocytosis. Some of the Schwann cells in lesions stained with anti-nitrotyrosine and anti-tumor necrosis factor-alpha antibodies. B. burgdorferi, or antigens thereof, were visualized immunohistochemically within macrophages. Forty-six months after infection, the most common changes were regenerative, whereas neuritis was infrequent. Aberrant axonal regeneration, irregularly sized myelinated fibers, and fibrosis were frequently observed. Possible mechanisms to explain the appearance and subsidence of Lyme neuritis are discussed.

A total of 46 Rhesus monkeys (RM) was inoculated with Mycobacterium leprae (ML) and followed clinically and immunologically for extended periods. Twenty-one (45.7%) of the RM developed leprosy spanning the known leprosy spectrum, with six of 21 (28.6%) having disease in the borderline lepromatous to lepromatous area of the spectrum. RM with paucibacillary forms of leprosy produced predominantly IgG anti-phenolic glycolipid (PGL-I) antibodies and positive lepromin skin test and/or in vitro blastogenesis responses; IgM anti-PGL-I predominated in animals with BB-LL leprosy and correlated with negative immune responses to lepromin. IgG anti-PGL-I antibodies persisted in a number of RM for several years without histopathological evidence of leprosy, suggesting possible persisting subclinical infection. The data show that RM are a valuable model for the study of leprosy. Eleven of the 46 RM were inoculated with ML from sources infected with simian immunodeficiency virus (SIV), the monkey counterpart to the human immunodeficiency virus (HIV). The possible effect of SIV on the clinical outcome of ML infection could not be determined due to insufficient numbers of animals to yield statistically significant results.

The efficacy of an outer surface protein A (OspA) vaccine in three different formulations was investigated in the rhesus monkey. The challenge infection was administered using Ixodes scapularis ticks that were infected with the B31 strain of Borrelia burgdorferi. Protection was assessed against both infection and disease, by a variety of procedures. Some of the animals were radically immune suppressed, as an attempt to reveal any putative low level infection in the vaccinated animals. The significant difference found between the spirochaetal infection rates of ticks that had fed on vaccinated vs. control monkeys, lack of seroconversion in the vaccinated animals, and the absence of spirochaetal DNA in the skin of vaccinated animals in the weeks following the challenge, indicate that vaccinated monkeys were protected against tick challenge. The post-mortem immunohistochemical and polymerase chain reaction analyses, however, suggest that these monkeys may have undergone a low-level infection that was transient.

Although there are several animal models of Lyme disease, only the rhesus monkey model exhibits all of the key manifestations of the disease. After infection with Borrelia burgdorferi, rhesus monkeys develop signs of early localized, early disseminated, and chronic Lyme disease. Specific features include erythema migrans, uveitis, myocarditis, arthritis, and disease of the peripheral and central nervous system. One of the unique features of the rhesus monkey model is the development of Lyme neuroborreliosis. Peripheral nervous system (PNS) involvement is usually in the form of a mononeuropathy multiplex with primarily axonal-loss features. Evidence of central nervous system (CNS) disease has included CSF pleocytosis, meningeal inflammation, spinal cord lesions, and polymerase chain reaction (PCR) data consistent with chronic CNS infection. The pathogenesis of Lyme neuroborreliosis is not well understood, but it is likely to involve complex interactions between B. burgdorferi and host immune mechanisms.

Peripheral neuropathy is a recognized but poorly understood manifestation of Lyme disease. We performed serial electrophysiological studies on 8 rhesus monkeys chronically infected with the JD1 strain of Borrelia burgdorferi and compared the results with those of similar studies on 10 uninfected control monkeys. Four infected and 2 uninfected animals underwent sural nerve biopsy. Five of the infected and 1 of the uninfected animals also had postmortem neuropathological examinations. Altogether, 5 of the infected monkeys demonstrated primarily axonal-loss-variety multifocal neuropathies. Only one nerve lesion exhibited findings compatible with demyelination. Pathologically, peripheral nerve specimens showed multifocal axonal degeneration and regeneration and occasional perivascular inflammatory cellular infiltrates without vessel wall necrosis. Free spirochetal structures were not seen, but several macrophages exhibited positive immunostaining with a highly specific anti-B. burgdorferi, 7.5-kd lipoprotein monoclonal antibody. In the infected animals, serial analysis of serum antibodies to B. burgdorferi showed increasing numbers of IgG specificities and new IgM specificities, suggesting persistent infection. Thus, peripheral neuropathy in the form of a mononeuropathy multiplex develops frequently in rhesus monkeys chronically infected with B. burgdorferi. The pathogenesis of these nerve lesions is not yet known, but our studies suggest an immune-mediated process perhaps driven by persistent infection with B. burgdorferi.

Background: We have previously reported the clinical, pathologic, and immunologic features of "early" Borrelia burgdorferi infection in rhesus monkeys (3). We have now evaluated these features during the chronic phase of Lyme disease in this animal model., Experimental Design: Clinical signs, and pathologic changes at the gross and microscopic levels, were investigated 6 months post-infection in several organ systems of five rhesus macaques (Macaca mulatta), which were infected with Borrelia burgdorferi by allowing infected Ixodes scapularis nymphal ticks to feed on them. A sixth animal was used as an uninfected control. Borrelia antigens recognized by serum antibody were identified longitudinally by Western blot analysis, and C1q-binding immune complexes were quantified. Localization of the spirochete in the tissues was achieved by immunohistochemistry and in vitro culture. The species of spirocheta cultured was confirmed by the polymerase chain reaction., Results: Chronic arthritis was observed in five out of five animals. The knee and elbow joints were the most consistently affected. Articular cartilage necrosis and/or degenerative arthropathy were the most severe joint structural changes. Synovial cell hyperplasia and a mononuclear/lymphocyte infiltrate were commonly seen. Nerve lesions were also observed, including nerve sheath fibrosis and focal demyelinization of the spinal cord. Peripheral neuropathy was observed in five out of five animals and could be correlated in the most severely affected monkey with the presence of higher levels of circulating immune complexes. Differences in disease severity did not correlate with differences in the antigens recognized on Western blot analysis., Conclusions: B. burgdorferi infection in rhesus macaques mirrors several aspects of both the early and chronic phases of the disease in humans. This animal model will facilitate the study of the pathogenesis of Lyme arthritis and neuroborreliosis.

Seven 72-hr-old Indian origin rhesus monkeys (Macaca mulatta) were inoculated with 10 animal ID50 of SIV/DeltaB670. Nine age-matched animals were used as uninoculated controls. All seven inoculated animals became infected as verified by viral isolation and SIV p26 antigenemia. Five of seven infected animals died within a mean of 31 days (range, 26-41 days), with high levels of antigenemia beginning 1-2 weeks postinoculation (PI) that persisted until death. Absolute lymphocyte numbers were within normal limits in all animals in both groups throughout the study. Inoculated animals that died within a mean of 31 days (short-term survivors) had significantly lower numbers of CD4+CD29+ (helper/inducer) lymphocytes than did long-term surviving inoculated animals through 3 weeks PI. Numbers of CD4+ lymphocytes were no different when controls were compared to all inoculated animals through 4-5 weeks PI. The two inoculated animals surviving 216 and 423 days PI (long-term survivors) did demonstrate declining CD4+ cells, but only late in disease. CD8+ lymphocytes were significantly lower in short-term survivors when compared to long-term survivors through 5 weeks PI. Antibody production against SIV viral proteins was detected only in the long-term survivors and was similar to results from past studies in juveniles. Clinical signs in the inoculated group were consistent with those seen in past studies on older animals. Persistent bacterial infections, primarily of the GI and respiratory tracts, were seen in the infected group. Aside from the lack of some opportunistic infections such as cytomegalovirus (CMV) and Pneumocystis carinii, necropsy findings were not different when compared to past studies on juvenile animals.(ABSTRACT TRUNCATED AT 250 WORDS)

We demonstrate that Borrelia burgdorferi infection in the rhesus monkey mimics the early and early disseminated phases of human Lyme disease. Clinical, bacteriological, immunological, and pathological signs of infection were investigated during 13 weeks after inoculation of the spirochete. Three animals were given B. burgdorferi (strain JD1) by needle inoculations, six animals were exposed to the bite of B. burgdorferi-infected Ixodes dammini ticks, and three animals were uninfected controls. B. burgdorferi could be recovered from all animals that were given the spirochete. Bacteria were detectable until week 6 postinoculation (p.i.) in blood, until week 8 p.i. in skin biopsies, and at 10 weeks p.i. in the conjunctiva of one of two animals which developed conjunctivitis. Erythema migrans (EM) appeared in one of the three animals infected by needle inoculation and in five of the six animals infected by ticks. Deep dermal perivascular lymphocytic infiltrations (characteristic of human EM) were observed in all animals showing EM clinically. Both EM and conjunctivitis were documented concomitantly with the presence of the spirochete. Lethargy, splenomegaly, and cerebrospinal fluid pleocytosis were also noted in some animals, but the direct connection of these signs with the infection was not shown. The appearance rate of immunoglobulin M and immunoglobulin G antibodies to B. burgdorferi, as well as the antigen spectra recognized, were remarkably similar to those seen in humans. Serum antibodies from infected animals were able to kill B. burgdorferi in vitro in the presence of rhesus complement. The rhesus monkey model appears to be useful for the investigation of the immunology and pathogenesis of Lyme disease and for the development of immunoprophylactic, diagnostic, and chemotherapeutic protocols.

Eight sooty mangabey monkeys were inoculated intravenously and intradermally with varying doses of Mycobacterium leprae from 4.8 x 10(7) to 4.8 x 10(10). Serum samples were obtained from the animals at intervals of about 3 months for 90 months, and were examined for IgM and IgG antibodies to nerve antigens, including ceramide, galactocerebroside (GC), and asialo-GM1 (AGM1), using an enzyme-linked immunosorbent assay (ELISA). The serological results were then compared with clinical findings, particularly nerve involvement. Of 8 mangabey monkeys inoculated with M. leprae, 7 animals had clinical leprosy; 6 of them had nerve damage, including neurologic deformities in 4 monkeys and nerve enlargement in 2. Median time for the initial signs of leprosy was 10 months postinoculation (p.i.), a range from 4 to 35 months. In contrast, nerve damage was noted rather late, about 35 to 86 months p.i. (median 54 months). The major immunoglobulin class to ceramide, GC, and AGM1 antigens was IgM, and the antibody responses to the nerve antigens appeared from 15 to 63 months p.i. (median 37 months). Antineural antibodies were thus detectable about 18 months (range -2 to 60 months) prior to observable nerve damage. In addition, elevation of antineural antibody levels were predictive of clinical exacerbation of the disease and neuritic damage. This study suggests that antineural antibodies are produced during the course of M. leprae infection and may be indicative of nerve damage, such as neurological deformities or nerve enlargement, in leprosy patients.