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Body Changes in HIV+ Monkeys
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"Body-Composition Changes in the Simian Immunodeficiency Virus--Infected Juvenile Rhesus Macaque"
Lisa M. Freeman,1 Keith G. Mansfield,2 Barry Goldin,3 Margo Woods,3 Lisa Gualtieri,3 Wenjun Li,3,4 Sarah Bussell,2 Andrew Lackner,2,a and Sherwood L. Gorbach3
1Department of Clinical Sciences, Tufts University School of Veterinary Medicine, North Grafton, 2Harvard Medical School, New England Regional Primate Research Center, Southborough, and 3Department of Community Health, Tufts University School of Medicine, and 4Biostatistics Research Center, Tufts--New England Medical Center, Boston, Massachusetts
ABSTRACT
Background. Body-composition changes are common in individuals infected with human immunodeficiency virus. The purpose of the present study was to measure, as a model of wasting in acquired immunodeficiency syndrome (AIDS), longitudinal body-composition changes in macaques infected with simian immunodeficiency virus (SIV).
Methods. Twelve juvenile macaques were inoculated with SIVmac239. Immunologic, virologic, somatometric, and dual-energy x-ray--absorptiometry measurements were performed prospectively every 4 weeks for 72 weeks and were compared to measurements taken from 8 uninfected control macaques.
Results. During the first 4 weeks, body-fat percentage decreased in the SIV-infected macaques while lean-tissue percentage increased; during weeks 4--72, these macaques lost a greater percentage of total fat tissue but had more subcutaneous-fat deposition than did the uninfected control macaques. Just prior to death, the SIV-infected macaques that died (n = 7) had a greater loss in body-mass index, abdominal fat, fat tissue, and lean tissue, compared with that in SIV-infected macaques that survived (n = 5).
Conclusions. Body-composition changes in SIV-infected juvenile macaques exhibit 3 phases: during acute infection, loss of body weight from fat tissue; a compensation period during which macaques grow, but at a reduced rate; and a terminal phase, during which tissue is lost from all body compartments. The SIV-infected juvenile macaque provides a useful model for the investigation of wasting in AIDS, particularly for pediatric AIDS wasting.
BACKGROUND
Body-composition changes in patients with AIDS are of considerable interest. Weight loss of >10% is extremely common in patients with AIDS and is, in fact, an AIDS-defining manifestation. Weight loss, however, may be a relatively insensitive measure of body-composition changes, because alterations within specific body compartments may precede overall weight loss. Loss of weight, especially of lean tissue, affects strength and immune function and is independently correlated with increased morbidity and mortality. Therefore, in patients with AIDS, measurement of the composition of specific body compartments—rather than just of body weight—may provide important and more-sensitive information.
Although information on body composition in patients with AIDS is important, the phenomenon is difficult to study in a controlled, longitudinal fashion. Several cross-sectional studies have shown that early weight loss occurs primarily in the fat compartment; then, once fat is considerably reduced, loss of lean body mass occurs. Another limitation of body-composition studies in patients with AIDS is that, in general, the period of early infection cannot be measured; therefore, most studies have evaluated the late stages of the disease. Finally, some drugs—particularly protease inhibitors—used in the treatment of AIDS alter body composition.
The simian immunodeficiency virus (SIV)--infected macaque model of AIDS has been used extensively to examine pathogenesis, vaccine strategies, and viral determinants of disease, because these lentiviruses closely parallel HIV. Disease manifestations resulting from infection with SIVmac239 include CD4+ depletion, giant-cell encephalitis (AIDS encephalitis), interstitial pneumonia, diarrhea, weight loss, malabsorption, and opportunistic infections. The clinical disease associated with SIV infection in rhesus monkeys is similar to the clinical syndrome in humans with AIDS, except that the disease course is greatly shortened in monkeys—a median survival time of 59 weeks makes possible a longitudinal study of the entire course of disease. In addition, controlled studies of the natural course of body-composition changes, from immediately after inoculation to the end of the disease, are possible.
In contrast to adult patients with AIDS, who exhibit weight loss according to their percentage of body fat prior to the weight loss, children with AIDS show a failure to gain weight according to age milestones (hereafter, "failure to thrive"). SIV-infected rhesus macaques that are in the growth stage provide a good model for children with AIDS. One study of juvenile rhesus macaques infected with SIV also demonstrated failure to thrive in terms of body weight. In that study, mean body weight at the time of death (mean, 74 weeks after inoculation) was 3.0 kg, whereas the published mean body weight for this age of male rhesus macaque is 4.1 kg. There also were significant differences between the growth pattern in SIV-infected rhesus macaques and that in uninfected rhesus macaques . Finally, body weight was inversely correlated with viral load, suggesting that failure to thrive is a consequence of SIV infection and may be related to the severity of infection.
Despite the information on body-weight changes in SIV-infected macaques, more-specific body-composition changes have not been documented in this model. Measurement of the fat compartment, lean-body-mass compartment, and bone compartment are important because they are more-sensitive indicators of body-composition changes, particularly in the growing juvenile (in which changes can be detected at an early stage). In addition, these measurements may provide additional information on the pathogenesis of these changes. Therefore, the purpose of the present study was to prospectively determine body-composition changes in SIV-infected juvenile rhesus macaques, compared with those in matched, healthy control rhesus macaques.
Animals. Twenty male juvenile rhesus macaques were studied; a group of 12 macaques were infected with SIVmac239 (50 ng of p27 viral-antigen equivalent, administered intravenously) and a group of 8 macaques were uninfected and used as healthy controls. The group of 12 macaques were infected (week 0) at a mean age ± SD of 68.8 ± 7.1 weeks, and both groups were followed for 72 weeks. All rhesus macaques were housed at the New England Regional Primate Research Center and were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. Clinical procedures were performed under the direction of a veterinarian, and all possible measures were taken to minimize discomfort in the rhesus macaques. Appropriate anesthesia and analgesics were used and were administered under the direction of a veterinarian; tiletamine was routinely used during handling of macaques. If the veterinary staff considered it to be necessary, rhesus macaques were killed in accordance with the recommendations of the American Veterinary Medical Association Panel on Euthanasia. Macaques were fed a certified commercial primate diet (Certified Primate Diet 5048; Purina Mills) ad libitum.
Body composition. Body weight was measured every 4 weeks, by use of a calibrated scale that measured to the nearest gram. For somatometrics, the following measurements were made by a single investigator (S.B.): crown-heel length (from the top of the unflexed head to the bottom of the heel), upper-arm circumference (at mid-biceps level), upper-leg circumference (at mid-quadriceps level), chest circumference (at the level of the nipples), and abdominal circumference (at the level of the umbilicus). A body-mass index was calculated as body weight (in kg) divided by the square of the crown-heel length (in cm).
Measurement of specific body compartments (fat, lean body mass, bone-mineral content, and bone-mineral density) was performed by dual-energy x-ray absorptiometry (DEXA) (Prodigy; GE Lunar), with software developed specifically for small primates. The software provided information on fat tissue and lean tissue, both in total grams and as a percentage of body weight; bone-mineral content, in grams; and bone-mineral density, in grams per square centimeter.
Ultrasound measurement of the thickness of abdominal subcutaneous fat was performed by ultrasonography (Hewlett-Packard Image Point, Phillips Medical Systems). With the macaque in dorsal recumbency, the thickness of the abdominal-fat layer was measured, in millimeters, on the midline, 1-cm cranial to the umbilicus, by use of a 7.5-MHz curvilinear transducer (Hewlett-Packard, Phillips Medical Systems).
RESULTS
In the group of infected macaques, 7 deaths due to SIV-related causes occurred, whereas, in the control group, no deaths occurred. The median survival time of the SIV-infected macaques was 67 weeks (range, 16 to >72 weeks). At baseline, there were no statistically significant differences, in either CD4+ and CD8+ T cell numbers or in body-composition measurements, between the group of SIV-infected macaques and the control group. However, 4 weeks after inoculation, the group of SIV-infected macaques experienced a statistically significant decline in CD4+ T cell count (P = .034), and the count remained at this lower level throughout the rest of the study.
In the group of SIV-infected macaques, a number of statistically significant body-composition changes occurred during the first 4 weeks after inoculation: body weight (P = .032) and chest (P = .030), abdominal (P = .021), and arm (P = .027) circumferences all decreased, as did the thickness of abdominal subcutaneous fat (P < .001). Although the absolute number of grams of body fat and lean body mass did not change, the percentage of fat decreased while the percentage of lean tissue increased (P < .001, for both). This group had no changes in bone-mineral content or bone-mineral density during the first 4 weeks after inoculation.
Both the SIV-infected macaques and the control macaques showed a nonlinear growth trend in body weight over time (P < .001), but the change in body weight was not different between the 2 groups. Both the control macaques and the SIV-infected macaques showed a linear increase in crown-heel length over time (slope, 0.945 [SE, 0.064] [P < .001] and slope, 0.507 [SE, 0.122] [P < .001], respectively). There was a statistically significant difference between the change in crown-heel length in infected macaques and that in control macaques (P = .017). Although, over time, there was an increase in the body-mass index for both the SIV-infected macaques and the control macaques (P < .001), there was no statistically significant difference between the indexes of the 2 groups (P = .139). In both the SIV-infected macaques and the control macaques, chest, abdominal, arm, and leg circumferences increased during the course of the study, but there was no statistically significant difference between the changes in the 2 groups.
A statistically significant decrease in fat tissue and a statistically significant increase in lean tissue (both on an absolute basis and as a percentage of total body composition [hereafter, "percentage basis"]) were detected, by DEXA, in the SIV-infected macaques 4--72 weeks after inoculation. The control macaques showed a statistically significant decrease in fat tissue on a percentage basis and a statistically significant increase in lean tissue on an absolute basis. There were statistically significant differences between the group of SIV-infected macaques and the group of control macaques, for both changes in fat tissue (both absolute and percentage basis) and changes in lean tissue (percentage basis) over time (table 1). Bone-mineral content and bone-mineral density increased in both the SIV-infected macaques and the control macaques (P < .001, for both), but there were no statistically significant differences between the changes in the 2 groups. The uninfected control macaques also experienced a statistically significant loss in abdominal subcutaneous fat, as measured by ultrasound (P = .035). In the SIV-infected macaques, the percentage of lean tissue was positively correlated with viral load (slope, 0.039 [SE, 0.014] [P = .004]), whereas the percentage of fat tissue (slope, -0.039 [SE, 0.019] [P = .036]) and bone-mineral content (slope, -0.127 [SE, 0.053] [P = .042]) were negatively correlated with viral load.
Terminal changes in the SIV-infected macaques that died (n = 7) were compared with changes in those that survived (n = 5) at a comparable time point. Compared with the SIV-infected macaques that survived, the SIV-infected macaques that died had statistically significant greater weight loss (P < .001) and were shorter (P < .001), resulting in a lower body-mass index (P = .009). The SIV-infected macaques that died experienced a greater decrease in chest (P = .007), abdominal (P = .007), arm (P < .001), and leg (P < .001) circumferences than did SIV-infected macaques that survived. Compared with the SIV-infected macaques that survived, the SIV-infected macaques that died also lost a statistically significant greater amount of abdominal fat (P < .001), as measured by ultrasound. Finally, compared with the SIV-infected macaques that survived, the SIV-infected macaques that died lost more fat tissue (absolute basis [P = .006]), lean tissue (absolute basis [P = .002]), and bone-mineral density (P = .020), as measured by DEXA. Although there was a statistically significant loss of tissue from all body compartments during the late stage of the disease, loss of body weight was due primarily to loss of lean tissue.
AUTHOR DISCUSSION
In our study of SIV-infected juvenile macaques, we observed 3 distinct phases: (1) acute infection, during which there was a loss of body weight, primarily from the loss of fat tissue; (2) a compensation period, during which there was growth, but at a reduced rate; and (3) a terminal period, during which there was loss of tissue from all body compartments. Longitudinal studies conducted from the time of infection until death have not been performed in humans, so we do not know whether these phases of body-composition changes also occur in people. One of the advantages of the SIV model is that acute body-composition changes that occur immediately after infection can be studied. In the present study, changes that occurred in SIV-infected macaques during the first 4 weeks included decreases in body weight; in chest, abdominal, and arm circumferences; and in abdominal-fat thickness. Also during this period, the percentage of body fat decreased while the percentage of lean tissue increased, suggesting that body-compartment changes began very early in the disease process and continued throughout the course of the disease; these early changes may negatively impact body composition throughout the course of the disease.
During weeks 4--72 of the present study, the SIV-infected juvenile rhesus macaques demonstrated stunted linear growth, compared with control macaques; this phenomenon is similar to the failure to thrive seen in children infected with HIV. This change in linear growth, despite the lack of difference in body weight, is a sensitive measure of the impact that SIV infection has on the juvenile host, because a failure to thrive is seen earlier than are changes in body weight. In terms of more-specific body-composition measurements, by DEXA, during the compensation period, the SIV-infected macaques experienced a preferential loss of fat but maintained lean tissue. The changes in fat tissue, lean tissue, and bone-mineral content were correlated with viral load. In humans, loss of fat also occurs early in the course of the progressive disease, and this is followed by loss of lean body mass as the fat mass is depleted. During growth in SIV-infected macaques, weight gain may continue to occur by the preferential depletion of fat tissue rather than lean tissue.
Although the SIV-infected macaques continued to grow (albeit at a reduced rate) throughout the present study, just before death they often exhibited wasting; therefore, the body composition of SIV-infected macaques just prior to death was compared with that of surviving SIV-infected macaques. During the period immediately preceding death, a number of changes were detected, including decreased body weight; shorter length; decreased body-mass index; smaller body circumferences; less fat tissue and lean tissue; and less abdominal fat. Thus, at this advanced stage of disease, it was no longer just that the growth of the macaques was stunted; they now exhibited marked wasting from all body compartments, and particularly from lean tissue. This is similar to what is seen in adult humans with AIDS.
Failure to thrive and wasting are often multifactorial problems that result from altered food intake, malabsorption, opportunistic infections, and increased production of inflammatory cytokines. Gastrointestinal absorption, energy expenditure, and cytokine production were not measured in the present study, although these parameters might help to clarify the reasons for the failure to thrive seen in the SIV-infected juvenile macaques.
The use of DEXA in this study allowed for the body-composition measurement of SIV-infected juvenile macaques. The measurement of specific body compartments (i.e., lean body mass, fat, and bone) allows a more-sensitive assessment of body-composition changes; small changes in weight may translate to more-important qualitative body-composition changes, such as in the lean-body-mass or bone compartments. The use of DEXA with these small monkeys, however, required the design of specific software for analysis. Standard software for use with pediatric subjects or small animals results in errors (e.g., nearly 20% overestimation of total mass of macaques); therefore, measurement of individual body compartments by these software programs may not produce accurate results.
Growth in juvenile macaques is a sensitive measure of the effects that SIV infection has on body composition, because deficits are seen early and because small changes in nutritional status can have large impacts on both overall growth and fat and lean-body-mass compartments. Additional studies are needed to both better characterize the weight loss in terms of specific body-composition changes and determine methods to either reverse or minimize these losses.
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