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Lipoatrophy & Mitochondrial Toxicity
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CROI, Session 144 - Lipoatrophy and Mitochondrial Dysfunction
Tuesday, February 27, 2007
Carl J. Fichtenbaum, M.D. University of Cincinnati College of Medicine
There were six papers presented in this session. Lipodystrophy is an important medical problem in HIV infected persons, particularly those taking antiretroviral therapy. Understanding the who is more likely to be affected is critically important. In the first abstract (800) by Hulgan and colleagues, they describe a polymorphism in the hemachromatosis gene is associated with greater likelihood of lipoatrophy. The study was done in patients randomized to nelfinavir or efavirenz based regimens in ACTG 384, a treatment na•ve study that included the use of d4T, ZDV and other nucleosides. In this subset of patients in a metabolic study, presence of the H63D heterozygous haplotype appeared to have a protective effect against lipoatrophy when controlling for other factors. The speculative mechanism may have something to do with iron metabolism and inflammation in adipose tissue. There were too few subjects with the homozygous haplotype to know if there is progressive effect. This interesting finding may lead to further mechanistic understanding of why some patients exposed to certain antiretrovirals develop lipoatrophy while others donÕt. In abstract 801, Stankov and colleagues demonstrated that thymidine analogs cause widespread mitochondrial damage in the mouse model (in multiple organs, brain, liver, heart, muscle, subcutaneous fat, and weight loss). This has been demonstrated by others in many model systems. It represents an avenue for research into understanding how antiretroviral therapy in the absence of HIV infection directly affects mitochondria and other tissues. In abstract 802, Nasi and colleagues found no difference in mitochondrial mtDNA levels of subcutaneous fat tissue at the time of delivery in HIV infected women exposed to various antiretrovirals compared to uninfected controls. The controls were seronegative women getting plastic surgery so it is not clear how comparable this is to someone who is pregnant. In addition, this really needs to be a longitudinal study comparing adipose tissue before and after exposure to know whether there are or are not changes associated with antiretroviral use. In abstract 803, Visnegarwala and colleagues from the SMART study reported on a subset of subjects who underwent more intensive metabolic assessments. Not surprisingly when stopped therapy, subjects had improvements in lipid levels. IÕm not convinced that fat was really gained in this study. Figure 1 shows no difference in fat between the arms from baseline to follow up. Figure 2 shows an increase in fat mass, however, that may reflect loss of lean body mass and a change in proportions. This is a distinct possibility since subjects off antiretroviral therapy may lose lean muscle mass with HIV progression. In any event, the harm associated with treatment interruption reported by the SMART study in the New England Journal of Medicine, November 30, 2006 issue outweighs any metabolic benefits at this point. In summary, this study supports the overwhelming data that antiretrovirals cause metabolic disturbances and that removal can reverse some changes. However, this is not a strategy that should be widely adopted because of the risks associated with treatment interruption no matter what the CD4+ count. In abstract 804 McGrath and colleagues from BMS reported no difference in body composition using atazanavir alone versus combined with ritonavir. This is an important issue that is hotly debated. The nucleoside backbone was d4T extended release tablet and 3TC. The sample size of nearly 100 subjects in each arm may not be sufficient to detect a clinical important difference, however. This type of analysis needs to be repeated in a prospective study to determine whether lower doses of ritonavir has an adverse effect. Further, these types of studies need to go longer than 48 weeks as the effect may be there but at a slower rate. In abstract 805, Mulligan and colleagues reported impressive improvements in visceral fat (reduced 30%) using Leptin (subcutaneously) without further loss of limb fat. There were also equally impressive improvements in lipid and glucose measures. This strategy might be useful for some patients. Currently, this medication is not available and requires twice daily dosing injections subcutaneously. It also sheds light on some of the potential mechanisms involved in metabolic disorders in persons with HIV. It is important to note that all of these patients had low leptin levels at the start. The majority of patients with lipoatrophy do not have low leptin so it is not clear what would happen if it was given in that setting. Finally, in abstract 806, Arenas-Pinto and colleagues reported on Lactate levels in 10 different Countries that were largely European sites. It is important to note in this case-control study that the controls were chosen by calendar year of entry and site to match to cases of persons with elevated lactate levels. 60% of controls had a CD4 count > 350 and were less likely, therefore to be exposed to antiretroviral therapy. I am not sure this is the most appropriate control group for these cases. What this tells me is that patients exposed to antiretrovirals are more likely to get elevated lactate levels and lactic acidosis, which is something that I think we already know. What is probably more helpful is to understand why some people exposed to specific medications get lactic acidosis and others do not.
800
The H63D Hemochromatosis (HFE) Gene Polymorphism and Mitochondrial Haplogroup J Are Associated with Limb Fat Changes after Initiation of ART: ACTG Study A5005s
Todd Hulgan*1, P Tebas2, J Canter1, K Mulligan3, D Haas1, M Dube4, S Grinspoon5, G Robbins5, A Kallianpur1, and ACTG Study 384 and A5005s Teams
1Vanderbilt Univ, Nashville, TN, US; 2Univ of Pennsylvania, Philadelphia, US; 3Univ of California, San Francisco, US; 4Indiana Univ Sch of Med, Indianapolis, US; and 5Massachusetts Gen Hosp, Harvard Univ, Boston, US
Background: Lipoatrophy can complicate ART and may be due in part to nucleoside reverse transcriptase inhibitor (NRTI) -induced mitochondrial dysfunction. Iron modulates both mitochondrial function and oxidative injury. In previous studies, NRTI-induced peripheral neuropathy was associated with European mitochondrial haplogroup T, and chromosomal HFE polymorphisms (C282Y and to a lesser extent H63D) were protective. Our objective was to evaluate the relationships between these genetic variants and body fat changes observed in HIV-infected individuals after the initiation of ART.
Methods: AIDS Clinical Trials Group (ACTG) 384 randomized ART-naive subjects to receive didanosine/stavudine or zidovudine/lamivudine in combination with efavirenz and/or nelfinavir. The metabolic substudy A5005s evaluated longitudinal changes in fat distribution by DEXA in a subset of patients at baseline and weeks 16, 32, 48, and 64. Subjects who also consented to participate in the ACTG Human DNA Repository (A5128) had HFE C282Y and H63D genotypes and mitochondrial haplogroups determined. Percentage change in total body, trunk, and limb fat at week 48 or 64 was compared by the Wilcoxon rank-sum test. We used logistic regression to determine whether these variants were associated with lipoatrophy (defined as a ³10% limb fat loss at week 48 or 64).
Results: Genetic data and either 48- or 64-week DEXA were available from 96 individuals (58% non-Hispanic white, 10% female). Overall median 48- or 64-week change in limb fat was -8.8% (IQR -28.7, +15.6). Among non-Hispanic white participants, 5 haplogroup J individuals had a 26.1% increase in limb fat at week 48 or 64 vs a 9.7% decrease in non-J individuals (n = 50, p = 0.07). Haplogroup T was present in 4 individuals and was not associated with fat changes. Among all subjects, HFE H63D heterozygotes (nucleotide 187 C>G; n = 23) had significantly less limb fat loss compared to homozygous wild type individuals (+6.1% vs -12.5%, p = 0.02) and were less likely to develop lipoatrophy (OR 0.31 [95%CI 0.10 to 0.95], p = 0.04) after adjustment for age, sex, race, and randomization arm. Conclusions: The common HFE H63D polymorphism was associated with protection from significant limb fat loss. There was also a trend toward protection in individuals belonging to mitochondrial haplogroup J. These results suggest that variation in iron metabolism genes and in mitochondrial genes may influence susceptibility to ART-associated lipoatrophy. These findings must be validated in larger studies.
801
Lipoatrophy and Ubiquitous mtDNA Depletion in Mice following Long-term Stavudine Treatment
Metodi Stankov*, R Schmidt, G Behrens, and German Competence Network HIV/AIDS
Hannover Med Sch, Germany
Background: Mitochondrial DNA (mtDNA) depletion has been proposed as an important factor leading to peripheral lipoatrophy in HIV-patients receiving ART. The extent to which mtDNA depletion occurs in other organs and tissue in humans has not been evaluated, and animal models for lipoatrophy have not yet been established.
Methods: Groups of mice were treated with stavudine (d4T), zidovudine (AZT), or vehicle (5 to 20 mice per group) for as long as 15 weeks with daily human doses adjusted for murine body surface area. To better parallel the pharmacokinetics in humans, drugs were administered via oral gavage. MtDNA contend was determined by real-time polymerase chain reaction (RT-PCR) in liver, muscle, heart, brain, and fat tissue. Adiponectin and leptin were measured by ELISA in the serum.
Results: Over a period of 15 weeks, mice receiving d4T or AZT gained less weight (d4T from 25.3±0.17 g to 29.1±0.2 g; AZT from 25.8±0.2 g to 30.2±0.2 g) than control animals (from 26.1±0.2 g to 34.8±0.2 g), while no differences in food and water intake were detected. Post mortem examination revealed that mice on AZT treatment, but particularly animals receiving d4T, had less of peripheral as well as central fat. Similarly, d4T-treated animals had lower serum adiponectin levels than control mice (2.62±0.1 µg/mL vs 3.05±0.1 µg/mL, p <0.05) and lower serum leptin concentrations (222.5±72.5 mg/mL vs 621.8±166 mg/mL). Analysis of mtDNA content revealed depletion of mtDNA in organs including the brain (-20%) and fat tissue (-27%), but the most profound and significant depletions were evident in muscle (-56%), liver (-64%), and heart (-45%).
Conclusions: This is the first study to show fat loss and hypoadiponectinema in mice after treatment with thymidine-analogs. Long-term treatment of mice with d4T led to mtDNA depletion that was not restricted to fat tissue. These data indicate that mtDNA depletion is an unspecific event during treatment with thymidine-analogs. If indeed mtDNA depletion is a major contributor to mitochondrial dysfunction, potential long-term toxicities in various organs must be considered.
802
Mitochondrial Toxicity in Adipose Tissue from HIV-infected Women during Pregnancy
Milena Nasi*1, E Chiesa2, G Rocco1, F Sabbatini1, A dÕArminio Monforte3, S Fiore4, E Ferrazzi4, N Palai5, C Campatelli6, and A Cossarizza1
1Univ of Modena and Reggio Emilia, Italy; 2Busto Arsizio Hosp, Varese, Italy; 3San Paolo Hosp, Milan, Italy; 4L Sacco Hosp, Milan, Italy; 5Univ of Brescia, Italy; and 6S Annunziata Hosp, Florence, Italy
Background: Increasing numbers of pregnant HIV+ women are receiving combination ART regimens for prevention of mother-to-child virus transmission or for treatment of the infection itself. Several studies have demonstrated that nucleoside reverse transcriptase inhibitors (NRTI) induce mitochondrial toxicity by several mechanisms, including depletion of mtDNA, whose levels are considered a marker of NRTI toxicity. By the quantification of mtDNA levels, we studied the role of mitochondrial toxicity in HIV+ women during pregnancy and the possible correlation with antiretroviral regimen, viro-immunological and metabolic parameters.
Methods: HIV+ women were enrolled in the framework of the Italian Prospective Cohort Trial on Efficacy and Toxicity of Antiretroviral in Pregnancy (TARGET study). We analyzed 36 patients (mean age 32.6 years; 55.5% Caucasian, 36.1% African American, 2.8% Hispanic, and 5.6% other), that were in A1 CDC class (25.0%), A2 (41.7%), A3 (25.0%), and C2 (8.3%). During pregnancy, 25 patients were treated with lamivudine (3TC), 23 with zidovudine (AZT), 9 with combivir (AZT+3TC), and 4 with abacavir (ABC); 2 received didanosine (ddI), 1 stavudine (d4T), 16 assumed nevirapine (NVP), 8 nelfinavir (NFV), 4 lopinavir/ritonavir (LPV/r), and 4 tenofovir (TDF). Median time of treatment during pregnancy was 164 days. At the third, sixth, and ninth month of pregnancy we collected data on: CD4 count, plasma HIV RNA, total and HDL-cholesterol, fasting glucose, and triglycerides. By real-time polymerase chain reaction (RT-PCR) (mtDNA qPCR), we quantified mtDNA copies per cell in subcutaneous fat samples collected during delivery. Statistical analysis was performed using Prism 3.03.
Results: The mean of mtDNA copies per cell were 452±198 (mean±SD, range 178 to 922). Such value is similar to that found in subcutaneous fat from 10 age- and sex-matched healthy subjects (378±229). We found no association between mtDNA and CD4 count during pregnancy, HIV RNA, use of protease inhibitor (PI) or NNRTI in association with NRTI, total and HDL-cholesterol, fasting glucose, and triglycerides. No correlation was found between therapy length and exposure to drugs. During pregnancy, total and HDL cholesterol and triglycerides increased significantly, while fasting glucose remained constant; no association with mtDNA levels was observed.
Conclusions: The therapy did not cause changes in adipose tissue mtDNA content, suggesting that treatment is apparently devoid of relevant mitochondrial toxicity.
803
The Effects of Intermittent, CD4-guided ART on Peripheral Limb Fat and Metabolic Parameters: The SMART Body Composition Substudy
Fehmida Visnegarwala*1,2, Fehmida Visnegarwala*1,2, B Grund3, A Thomas3, K Ellis1, C Gibert4, J Shlay5, F Drummond6, E Martinez7, W El-Sadr8, A Carr9, and Body Composition Sub-study Investigators of the SMART trial
1Baylor Coll of Med, Houston, TX, US; 2CPCRA; 3Univ of Minnesota, Minneapolis, US; 4VAMC, Washington, DC, US; 5Denver CPCRA, Denver Publ Hlth, Univ of Colorado Hlth Sci Ctr, US; 6Natl Ctr for HIV Epidemiology and Clin Res, Univ of New South Wales, Australia; 7Hosp Clin, Univ of Barcelona, Spain; 8Harlem Hosp, Columbia Univ, New York, NY, US; and 9St Vincent's Hosp, Univ New South Wales, Sydney, Australia
Background: Interruption of ART has been used in an attempt to mitigate its body fat and atherogenic metabolic abnormalities. This has not been assessed in a randomized trial. SMART randomized patients with CD4 counts >350 cells/mm3 to intermittent, CD4-guided ART (stop ART >350 and start <250 cells/mm3) (drug conservation) or continuous ART (viral suppression).
Methods: Participants from 32 sites in the United States, Australia, and Spain were co-enrolled in a body composition substudy. A key primary aim was to evaluate peripheral limb fat mass and limb fat percent by annual DEXA. Fasting (³12 hours) lipid and glycemic parameters were done at months 4 and 8 and annually. Statistical comparisons of randomized groups were adjusted for sex, race, and baseline values.
Results: We randomized 142 and 133 patients to the drug conservation, and viral suppression arms, respectively. At baseline, mean age was 48 years, 19% were female, 26% were black, 22% had prior AIDS, 39% had provider-reported lipoatrophy, 92% were ART-experienced (74% on ART at baseline), and 8% na•ve; median CD4 count was 562 cells/mm3 and 58% had HIV RNA ²400 copies/mL. Median baseline limb fat mass was 6.6 kg (limb fat 8.4%), and mean total cholesterol was 190 mg/dL, HDL-C 40 mg/dL, LDL-C 110 mg/dL, and triglycerides 226 mg/dL. At 12 months, 45% of patients in drug conservation had re-initiated ART. After 12 months, mean limb fat mass changes were +0.12 kg in drug conservation and -0.14 kg in viral suppression (estimated difference 0.32 kg, 95%CI -0.19 to 0.85, p = 0.22, n = 213 patients), mean changes of 7.6% and 0.5%, respectively (estimated difference 7.4%, 1.5 to 13.3%, p = 0.02). Mean changes in limb fat percent were +0.2% and -0.2%, respectively (estimated difference, 0.5%, 0.0 to 0.9%, p = 0.03). Treatment difference in limb fat change was similar for those on or off ART at baseline (p >0.4 for interaction, all 3 outcomes). There were significant reductions in lipids in drug conservation relative to viral suppression starting at month 4. Month 4 lipid changes were (n = 165 patients): total cholesterol (estimated difference -36 mg/dL, -48 to -24, p <0.001), LDL-C (estimated difference -16.9 mg/dL, -26.0 to -7.8, p <0.001); HDL-C (-5.1 mg/dL, -8.0 to -2.1; p <0.001); triglycerides (-68 mg/dL, -101 to -35, p <0.001). There was no significant difference observed (n = 159) for glucose, insulin or C-peptide at month 4.
Conclusions: Compared to continuous ART, use of intermittent, CD4-guided ART modestly improved peripheral fat at 12 months, and resulted in lower fasting lipid, but not glycemic, levels over 4 months.
804
Body Composition Changes in ART-naive Subjects Treated with Atazanavir or Atazanavir/Ritonavir-based Once-daily HAART: 48-Week Computed Tomography and DEXA Data
Donnie McGrath*1, D Frederick1, V Wirtz1, R Yang1, A Thiry1, M Mathew1, and M Noor2
1Pharma Res Inst, Bristol-Myers Squibb, Wallingford, CT, US and 2Pharma Res Inst, Bristol-Myers Squibb, Princeton, NJ, US
Background: Prospective studies have shown that body fat changes in ART-naive patients treated with atazanavir (ATV) -based HAART are similar to an efavirenz (EFV) -based regimen at 48 weeks. However, the effect of the addition of ritonavir (RTV) to ATV on body composition is unknown.
Methods: This was a randomized trial in ART-naive HIV+ subjects (n = 200) who initiated ATV 400 mg or ATV/RTV 300/100 mg once daily, each with extended-release stavudine (d4T) and lamivudine (3TC) once daily. Body composition was assessed by computed tomography and DEXA imaging; in addition, lipodystrophy case definition scores were recorded using the validated FRAM questionnaire (subject/investigator reports). These parameters, plus body mass index and hip/waist measurements, were assessed at baseline, week 24 and week 48 (plus week 12 for the FRAM questionnaire). Comparisons of computed tomography and DEXA parameters used t-tests.
Results: Subjects on each regimen had comparable mean weight changes at 48 weeks from baseline (ATV 5%; ATV/RTV 6% ). Body mass index and waist circumference changes were comparable between regimens. There were minimal changes in waist-to-hip ratio. There were no statistically significant differences between regimens for body composition parameters. Loss of arm fat was reported at week 48 by 8% and 6% of the ATV and ATV/RTV subjects, respectively. Investigator-reported lipoatrophy rates at baseline and at week 48 were 10% and 4% on ATV and 6% and 5% on ATV/RTV; the rates for lipohypertrophy were 8% and 13% on ATV, and 5% and 11% on ATV/RTV.
Conclusions: There were no clinically significant differences in body composition between ATV and ATV/RTV in these d4T-containing regimens at 48 weeks based on computed tomography and DEXA imaging, and a validated questionnaire. The increases in trunk fat, visceral adipose tissue, and subcutaneous adipose tissue with both regimens are consistent with immune reconstitution and the return to health seen in the first year of HAART. The addition of RTV to ATV does not appear to be associated with clinically significant changes in body composition at 48 weeks.
805
Improvements in Hepatic and Adipocyte Insulin Sensitivity, Dyslipidemia, and Visceral Fat during Leptin Treatment in HIV-infected Men with Lipoatrophy and Hypoleptinemia
Kathleen Mulligan*1, H Khatami1, J M Schwarz1,2, J M Schwarz1,2, G Sakkas1, A DePaoli3, G Lee1, V Tai1, M Wen1, C Grunfeld1, and M Schambelan1
1Univ of California, San Francisco, US; 2Touro Univ, Vallejo, CA, US; and 3Amgen, Inc, Thousand Oaks, CA, US
Background: Leptin treatment improves lipid and glucose metabolism in non-HIV lipodystrophy. The present study was designed to determine whether leptin could produce similar benefits in HIV-infected patients with lipoatrophy.
Methods: In this open-label, proof-of-principle study, 8 HIV± men with lipoatrophy (with or without fat accumulation), hypoleptinemia (<3 ng/mL), dyslipidemia (despite use of lipid lowering agents in 7 of 8), and insulin resistance, received recombinant human leptin for 6 months (0.01 mg/kg twice daily for 3 months, followed by 0.03 mg/kg twice daily for 3 months). Glucose and lipid metabolism was assessed under fasting conditions and during a euglycemic, hyperinsulinemic clamp with stable isotope infusions before and after 6 months of treatment. Visceral and subcutaneous abdominal fat (at L4-L5) were measured by magnetic resonance imaging and peripheral fat by DEXA. Data are mean ±SE. Results were analyzed by paired t-test.
Results: There were significant decreases in fasting levels of total (229±16 to 187±12 mg/dL, p = 0.001), direct LDL (140±8 to 117±8 mg/dL, p = 0.002), and non-HDL (204±15 to 158±12 mg/dL, p = 0.001) cholesterol. HDL cholesterol tended to increase (25±2 to 29±3 mg/dL, p = 0.07). Triglyceride levels decreased significantly under fasting, hyperinsulinemic (clamp), and postprandial conditions. Whole-body lipolysis and free fatty acid levels decreased significantly during both fasting and hyperinsulinemia. Fasting insulin (21±3 to 16±2 mIU/mL, p = 0.04), endogenous glucose production (2.8±0.1 to 2.5±0.1 mg/kg/minutes, p = 0.005), and glycogenolysis (2.2±0.1 to 1.9±0.1 mg/kg/minutes, p = 0.03) decreased significantly, as did glucose production, glycogenolysis, and gluconeogenesis measured during the clamp, providing evidence of improvements in hepatic insulin sensitivity. Peripheral glucose uptake (clamp) improved modestly but the increase was not statistically significant. Visceral abdominal fat decreased by 30% (183±24 to 129±24 cm2, p = 0.001) with no significant changes in subcutaneous or peripheral fat. Leptin was well tolerated.
Conclusions: Leptin treatment was associated with marked improvement in dyslipidemia in a group of patients already treated with lipid-lowering agents. Visceral fat decreased with no exacerbation of peripheral lipoatrophy. Hepatic insulin sensitivity improved and lipolysis decreased. Results from this pilot study suggest that leptin may have therapeutic potential in HIV-infected patients with metabolic and morphologic alterations.
806
International Case-control Study of Lactic Acidosis and Severe Hyperlactatemia in Treated HIV-infected Adults
Alejandro Arenas-Pinto and International Lactic Acidosis Study Group
Royal Free and Univ Coll London Med Sch, UK
Background: Lactic acidosis and severe hyperlactatemia are rare but serious complications of ART. To identify risk factors for hyperlactatemia/lactic acidosis among HIV-infected adults, we conducted a case-control study, including 19 centers in 11 countries in Europe, the Americas, and Australia.
Methods: Lactic acidosis was defined as arterial blood pH <7.35, bicarbonate <20 mmol/L, and lactate above normal; hyperlactatemia was defined as 2 consecutive blood lactate readings >5 mmol/L, irrespective of acid-base status. We randomly selected 2 controls per case from patients >16 years, matched by center and calendar year. Conditional logistic regression was used to identify risk factors.
Results: Between 1997 and 2005, 110 cases (49 with lactic acidosis) were diagnosed and 220 controls were included: 36.4% of cases and 18.2% of controls were women (p <0.001); median age was 42.4 years for cases (inter-quartile range [IQR] 36.0 to 52.5) and 40 for controls (IQR 35.0 to 47.1) (p = 0.013); 41.8% of cases and 30.8% of controls were non-white (p = 0.014). In the univariate analysis cases were more likely to be receiving stavudine (d4T) (odds ratio [OR] 3.67, 95% confidence interval [CI] 2.20 to 6.14) or didanosine (ddI) (OR 5.54, 95%CI 3.04 to 10.10) at the time of the event, whereas controls were more likely to be exposed to zidovudine (AZT) or lamivudine (3TC). An even stronger association was exposure to a d4T/ddI-based combination (OR 25.31, 95%CI 7.43 to 86.23) compared with AZT/3TC-based combination. Case status was associated with shorter duration of exposure to both dideoxynucleosides. In a multivariable model adjusting for age, gender, and current CD4 count, hyperlactatemia and lactic acidosis remained associated with shorter duration on ddI (adjusted OR 8.39, 4.54 and 3.09 for patients exposed for <12, 12 to 24, and >24 months, respectively). In addition, age >40 years (adjusted OR 2.6, 95%CI 1.08 to 6.29), female gender (adjusted OR 5.97; 95%CI 1.92 to 18.5), and advanced HIV-induced immunosuppression are independently associated with hyperlactatemia and lactic acidosis (adjusted OR 5.66, 9.53 and 7.36 for patients with 200 to 349, 100 to 199, and <100 CD4, respectively).
Conclusions: Lactic acidosis and hyperlactatemia were strongly associated with exposure to dideoxynucleosides. The additional associations with female gender, advanced immunosuppression, and possibly ethnicity have important consequences for choice of ART regimens in developing countries. The association with shorter duration of exposure argues against a mechanism depending on cumulative exposure and may support the hypothesis of a particular susceptibility in some patients.
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