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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3817-3824
Effect of L-Carnitine on Human Immunodeficiency Virus-1
Infection-Associated Apoptosis: A Pilot Study
By
Sonia Moretti,
Edoardo Alesse,
Luisa Di Marzio,
Francesca Zazzeroni,
Barbara Ruggeri,
Sonia Marcellini,
Giuseppe Famularo,
Seth
M. Steinberg,
Antonio Boschini,
M. Grazia Cifone, and
Claudio De Simone
From the Department of Infectious Diseases, University La Sapienza,
Rome, Italy; the Department of Experimental Medicine, University of
L'Aquila, L'Aquila, Italy; Centro Medico di San Patrignano, Rimini,
Italy; and the National Cancer Institute, National Institutes of
Health, Bethesda, MD.
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ABSTRACT |
The Fas/Fas ligand system is involved in uncontrolled apoptosis,
which ultimately leads to the loss of T lymphocytes in human immunodeficiency virus (HIV)-infected individuals. The signal transduced by Fas receptor involves the activation of an acidic sphingomyelinase, sphingomyelin breakdown, and ceramide production. Our
recent reports have shown that L-carnitine inhibits Fas-induced apoptosis and ceramide production both in vitro and in vivo. The aim of
this study was to study, in a preliminary fashion, the impact of
long-term L-carnitine administration on CD4 and CD8 absolute counts,
rate, and apoptosis in HIV-1-infected subjects. The generation of
cell-associated ceramide and HIV-1 viremia was also investigated.
Eleven, asymptomatic, HIV-1-infected subjects, who refused any
antiretroviral treatment despite experiencing a progressive decline of
CD4 counts, were treated with daily infusions of L-carnitine (6 g) for
4 months. Immunologic and virologic measures and safety were monitored
at the start of the treatment and then on days 15, 30, 90, and 150. L-carnitine therapy resulted in an increase of absolute CD4 counts,
which was statistically significant on day 90 and 150 (P = .010 and P = .019, respectively). A positive, not significant
trend was also observed even in the change in absolute counts of CD8
lymphocytes. L-carnitine therapy also led to a drop in the frequency of
apoptotic CD4 and CD8 lymphocytes. This reduction occurred gradually,
but changes in actual values between each time point and baseline were
strongly significant (P = .001 at the end of the study
compared with the baseline). A strong reduction (P = .001) in
cell-associated ceramide levels was found at the end of the study. In
general, HIV-1 viremia increased slightly. No toxicity related to
L-carnitine therapy was observed and dose reductions were not
necessary. In HIV-1-infected subjects, long-term infusions of
L-carnitine produced substantial increases in the rate and absolute
counts of CD4 and, to a lesser degree, of CD8 lymphocytes. This was
paralleled by a reduced frequency of apoptotic cells of both subgroups
and a decline in the levels of ceramide. No clinically relevant change
of HIV-1 viremia was observed.
| |
INTRODUCTION |
LYMPHOCYTES FROM subjects infected with
the human immunodeficiency virus type 1 (HIV-1) undergo an
inappropriate programmed cell death (apoptosis), a major mechanism for
the decline of CD4 and CD8 cells that is crucial to the progression
towards the overt acquired immunodeficiency syndrome
(AIDS).1-8 Indeed, lymphocyte apoptosis correlates with
disease progression and lower CD4 counts: a high degree of apoptosis
has been detected in patients with AIDS in comparison with long-term
nonprogressors.1-8
Recent studies have provided evidence that the Fas-Fas ligand (FasL)
system is involved in the molecular mechanism of T-cell depletion in
HIV-1 infection and AIDS.9-12 Thus, viral gene products, such as gp120 and HIV-1 Tat, have been shown to greatly accelerate apoptotic death in T lymphocytes after the T-cell receptor triggering that is mediated via the Fas-FasL interaction. Furthermore, an increased expression of Fas on peripheral T cells from patients with
HIV-1 infection has been found that correlates with disease progression, CD4 cell depletion, and a strong increase in sensitivity of patient T cells for Fas-mediated apoptosis ex vivo.9-12
The apoptotic signal transduced by Fas receptor involves the activation of an acidic sphingomyelinase, sphingomyelin breakdown, and ceramide production.13,14 Ceramide acts as an endogenous mediator of apoptosis in several cell lines13-17 and also enhances
HIV-1 replication.18,19 Patients with AIDS have
significantly higher lymphocyte-associated ceramide levels than healthy
individuals and HIV-1-infected long-term nonprogressors have less
elevated lymphocyte-associated ceramide levels than subjects with
evolving disease.8,20,21 Remarkably, this is paralleled by
a lower frequency of apoptotic CD4 and CD8 cells in long-term
nonprogressors than in patients with AIDS.8,21
In a preliminary, clinical study, short-term treatment of AIDS patients
with L-carnitine resulted in a substantial reduction in the frequency
of apoptotic CD4 and CD8 cells, paralleled by reduced levels of
cell-associated ceramide.22 Of note, L-carnitine did not
seem to influence viral replication directly (H. Mitsuya, NCI, personal communication, May 1995). These in vivo
results were supported by in vitro studies. In fact, experiments
conducted using the Fas-sensitive HuT78 and U937 cell lines have shown
that L-carnitine inhibits both apoptosis and ceramide production
triggered by Fas cross-linking, and this appears to involve the
inhibition of an acidic sphingomyelinase.23 By contrast,
the activity of a Fas-activated neutral sphingomyelinase, which does
not seem to be implicated in the generation of ceramide, relevant to
Fas-induced apoptosis, is not significantly influenced by
L-carnitine.23
Taking everything into consideration, it is reasonable to deduce that
therapies directed at downmodulating the generation of ceramide may
slow the progression of HIV-1 infection by reducing apoptotic
lymphocyte death, even if unable to affect viral replication directly.
This background prompted us to investigate the impact of long-term
treatment, with a highly purified L-carnitine preparation, on absolute
counts, rate, and apoptosis in both CD4 and CD8 lymphocytes, ceramide
generation, and HIV-1 viremia. To perform this pilot trial, we enrolled
HIV-infected individuals who fulfilled the following eligibility
criteria: (1) each subject had to be living in the community of San
Patrignano,24 which is devoted to the rescue of drug
addicts and, (2) each individual had rejected the opportunity of
antiretroviral treatment despite experiencing a progressive decline in
CD4 cell counts.
| |
MATERIALS AND METHODS |
Study site.
San Patrignano is a private residential community for the
rehabilitation of drug users, located in northern Italy (Corione, Rimini, Italy24). The San Patrignano Community was
established in 1979 for individuals wishing to undergo rehabilitation.
The physical property, extending for 1,000 acres, consists of several residential buildings and extensive pastural and agricultural lands.
Treatment, which is free of charge, lasts for a mean time of 3 years.
The community rules prohibit the use of illicit or psychotropic drugs;
most all of the guests (>95%) are smokers, with a maximum of 10 cigarettes a day allowed. A maximum amount of one glass of wine a meal
was also allowed. To ensure abstinence from drug use, guests are not
allowed to leave the community during the first year of residency.
Since 1980 more than 10,000 drug addicts have been treated, 2,200 with
HIV infection.
Study population.
In accordance with the San Patrignano Medical Center protocol, all
newly entering drug users undergo the following examinations: physical
examination, chest x-ray, electrocardiogram, intradermal purified
proteic derivative from M. tuberculosis (PPD; 5 IU), delayed skin tests (multitest Merieux), and routine blood analyses, including serology for HIV, hepatitis B virus (HBV), hepatitis C virus
(HCV), and syphilis. HIV-infected individuals are further investigated for lymphocyte subsets, serology for human T-cell lymphotropic virus type I (HTLV-I), Toxoplasma
gondii, and cytomegalovirus; abdominal echography is also
performed. At least once a year, a serum and plasma sample from each
guest is obtained and stored, respectively at  20°C and
80°C.
For any type of medical problem, guests were sent to the Community
Medical Center, which includes an out-patient facility, a day-hospital
unit (16 beds), and a 36-bed ward. Chemoprophylaxis for HIV-1-related
opportunistic infections is administered after standard criteria. In
autumn, influenza vaccination is offered to all guests, with a mean
adhesion rate of 50%. Antiretroviral therapy is offered according to
the National Institutes of Health guidelines.
Patients and L-carnitine treatment.
Eleven, asymptomatic, HIV-1-infected, male subjects living in the
Community of San Patrignano (mean age, 33 ± 6.1 years; range, 27 to
43; mean CD4 cell counts, 305 ± 133 cells/mL; range, 61 to 558) gave written informed consent to participate in this pilot study. The study design was approved by the Internal Ethical Committee of S. Patrignano taking into account that the compound is freely available in Italy and had no untoward effects. The
patients were chosen among those with CD4 lymphocyte counts between 200 to 500/µL; those with CD4 >500 were not considered because they
were stable; those with CD4 <200 were not considered because they
were rapid progressors. The patients had been staying a
mean of 51 ± 4 months (range, 19 to 96) at the Community of San
Patrignano24 and had stable CD4 counts until when, over the
last 12 months of follow-up, progressively declining counts were shown.
Despite this, all patients freely refused to undergo antiretroviral
therapy with zidovudine and/or didanosine and were selected on
their willingness to be treated with L-carnitine. Obviously, it was
made clear to the patients that L-carnitine treatment did not take the
place of antiretroviral therapy. All subjects had stable body weight,
or maintained their weight within a 10% variation range during the last 3 months and had albumin levels above 4 g/dL and total carnitine levels comparable to those in healthy individuals. None had persistent or severe diarrhea in the previous 3 months and nutritional support was
not requested. None of these subjects had clinical or laboratory evidence of kidney dysfunction and the Karnofsky score was >90 in all
subjects. Notably, all of the individuals were HCV positive, with the
exception of patients no. 6684 (HBV positive) and 6457 (negative).
Patient no. 6438 was both HBV and HCV positive and patients no. 6466 and 2000 had a past episode of Herpes zoster infection.
The patients were administered 6 g/day highly purified L-carnitine
(Carnitor, Sigma Tau, Pomezia, Italy), intravenously, in normal saline
over a 2-hour period each day for 4 months. During the trial of
L-carnitine therapy, a complete physical examination was performed and
both blood counts and a biochemical profile were obtained at baseline
and then regularly each week throughout the entire study period. No
opportunistic infection intercurred during the present trial. Patients
were ranked on the Karnofsky performance score by the same investigator
on each visit over the course of the trial. To evaluate the frequency
of apoptotic CD4 and CD8 cells and the levels of peripheral blood
mononuclear cell (PBMC)-associated ceramide, blood samples were taken
at baseline (T0), on day 15 (T1), on day 30 (T2), on day 90 (T3), and
on day 150 (T4). HIV-1 viremia was measured at T0 and T4. Blood samples were taken at least 18 hours after the previous infusion of L-carnitine taking into account that the half-life of L-carnitine after intravenous infusion is less than 1 hour and that plasma concentrations return to
base level within 10 hours.25
PBMC isolation.
PBMCs were separated from heparinized peripheral blood by Lymphoprep
gradient centrifugation (Nicodem, Oslo, Norway), washed twice with
phosphate-buffered saline (PBS), incubated for 8 hours in RPMI 1640 supplemented with 5% fetal calf serum (FCS), L-glutamine, HEPES, and antibiotics at 37°C, and then used for cytofluorometric analysis of phenotype and apoptosis and extracted for ceramide level
determination.
Phenotypic analysis of apoptotic cells.
The absolute counts of cells bearing the CD4 or CD8 phenotype were
determined by flow cytometry. In brief, PBMCs were stained with an
anti-CD4 and an anti-CD8 monoclonal antibody labeled with fluorescein,
according to the manufacturer's instructions (Becton Dickinson
Immunocytometry Systems [BDIS], San Jose, CA).
Quantification and phenotypic analysis of apoptotic cells in
lymphocytes was performed by staining apoptotic cells with
7-amino-actinomycin D (7-AAD) (Sigma Chemical Co, St Louis, MO) as
prevously described.26 This method discriminated between
early and late apoptotic cells, evaluating the increase of 7-AAD
fluorescence of apoptotic cells due to the alteration of their membrane
permeability. Briefly, for the staining of apoptotic and dead cells,
PBMCs were incubated with 20 µg/mL of 7-AAD in 100 µL of PBS
(without Ca2+ and Mg2+, Irvine, Santa Ana, CA)
containing 2% newborn calf serum (Irvine) and 0.1% sodium azide
(Sigma) (PBSAz) for 20 minutes at 4°C protected from light; the
cells were then analyzed in their staining solution by flow cytometry
analysis. For staining of surface antigens, 10 µL of each anti-CD4
and anti-CD8 fluorescein isothiocyanate (FITC) monoclonal antibodies
(Becton Dickinson) in 100 µL of PBSAz were added to 1 × 106 cells followed by incubation for 15 minutes at 4°C.
For determination of background staining, cells were incubated with 10 µL each of mouse IgG1 FITC (Becton Dickinson). After one wash with 2 mL of PBSAz, the supernatant was removed and the cell pellet was
resuspended in 1 mL of PBSAz. The spectral properties of 7-AAD allow
the staining of apoptotic cells by fluorescence emission in the red
channel FL-3 (650 nm < wavelenght < 850 nm) and the easily and
simultaneous labeling of cell surface antigens (FL1 and FL2).
Identification of apoptotic cells was assessed also by considering
scatter characteristics, based on the evidence that apoptotic cells can
easily be distinguished from viable cells via measurement of forward
scatter (FSC) and side scatter (SSC) light parameters, which are
proportional, respectively, to cell diameter and internal granularity.27 Indeed, while living cells display
relatively high FSC/low SSC properties, cells undergoing programmed
cell death shift to a lower FSC/higher SSC compartment, consistent with
the cellular changes occurring during apoptosis (reduction of cell size
and cytoplasmic volume and chromatin
condensation).27,28
Ceramide measurement (diacylglycerol kinase assay).
PBMCs (2 × 106 cells), isolated as above,
were pelleted and resuspended in ice cold methanol:chloroform:water
(2.5:1.25:1). Incubation was stopped by immersion of samples in
methanol/dry ice (70°C) for 10 seconds followed by
centrifugation at 4°C in a microfuge. Lipids were extracted and
then incubated with Escherichia coli diacylglycerol (DAG)
kinase (DAG kinase assay kit and 32P-adenosine triphosphate
[ATP]  [specific activity 3 Ci/mmol/L], from Amersham,
Buckinghamshire, UK). Ceramide phosphate was then isolated by thin
layer chromatography using
CHCl3/CH3OH/CH3COOH (65/15/5,
vol/vol/vol) as solvent. Authentic ceramide (type III; from bovine
brain; Sigma) was identified by autoradiography at retention factor
(Rf) 0.25. Specific radioactivity of
ceramide-1-phosphate was determined by scintillation counting of
corresponding spots scraped off the gel. Quantitative results for
ceramide production were obtained from comparing the experimental
values with a linear curve of the ceramide standards and are expressed
as pmoles of ceramide-1-phosphate/106 cells.
Plasma polymerase chain reaction (PCR) for HIV-1 RNA determination.
Peripheral blood was collected from each subject in tubes containing
acid-citrate dextrose and processed within 6 hours. Plasma was
separated from whole blood by centrifugation and stored in liquid
nitrogen. We used 200 µL of plasma to estimate the HIV-1 RNA copies
per milliliter. The application of quantitative PCR was performed using
the Amplicor detection system (Roche, Branchburg, NJ) and the gene AMP
9600 thermal cycler (Perkin Elmer, Norwalk, CT). Shortly, the HIV-1 RNA
was extracted from each sample using guanidinium, reverse transcribed,
and amplified by recombinant thermus thermophilus (rTth)
DNA polymerase and detected using a method based on changes in optical
density produced by reactions mediated by horseradish peroxidase. A
noncompetitive, internal control was introduced to monitor sample and
reaction interference.
Statistical analysis.
For each of the several parameters of interest, differences were
calculated from each later time point and T0, and these differences tested against a null hypothesis of no change by using the Wilcoxon signed rank test; individual two-tailed P values are reported and the Hochberg adjustment for multiple comparisons was also used to
correct for analysis of four changes from baseline for each
parameter.29 For HIV-1 viremia, the logs of the ratios of
values at T0 and T4 were tested using the Wilcoxon signed rank test.
Spearman rank correlation was performed between ceramide levels and
apoptotic CD4 and CD8 cells on each of the time points throughout the
study period.
| |
RESULTS |
CD4 counts.
The absolute CD4 cell counts increased during the period when
L-carnitine was administered (Table 1).
There was a significant increase in CD4 counts at T3 and T4 compared
with T0 (P = .010 and P = .019), but a positive trend
was already evident at T1 and T2, even though the differences were not
statistically significant in comparison with the baseline values
(P = .56 for T1 v T0 and P = .52 for T2
v T0) (Table 2). The mean net
change from baseline at the end of the study was an increase of 101.09 ± 114.71 (range, 60 to +337 cells/µL). Two patients had an
increase of more than 100%, two patients more than 50%, and three
patients approximately 30% over the baseline CD4 cell counts. In three
patients, the treatment with L-carnitine did not result in a
significant gain of CD4 cells and a slight decrease was observed at the
end of the study compared with the baseline. However, in one of these subjects, slightly increased counts were observed at T2 and in another
of these subjects on T3.
CD8 counts.
At each time point over the period when L-carnitine was administered,
we observed a trend towards increased absolute CD8 cell counts, but the
impact of L-carnitine therapy did not result in statistically
significant gain (Table 1). The net change from baseline to the end of
the study was an increase of 164.36 ± 569.18 (range, 372 to
+1,585 cells/µL). At the end of the study, six patients had increased
counts (range, 154 to 1585) (Table 2). In particular, one patient had
an increase of more than 100%, three patients more than 50%, and two
patients 30%. The remaining five patients had a slight decline (range,
123 to 372) compared with the baseline.
Apoptotic CD4 and CD8 cells.
The frequency of apoptosis was investigated using the 7-AAD staining
method that allows the detection of apoptotic cells, which are stained
by 7-AAD because of their altered membrane integrity in contrast to
living cells. Using this method, L-carnitine therapy led to a
significant drop in the frequency of apoptotic CD4 and CD8 lymphocytes
compared with the baseline (Fig 1 and
2, respectively). This reduction
occurred gradually, but changes in actual values between each time
point and T0 were always strongly significant (P values were
between .001 and .014) (Table 2). The mean net change from baseline at
the end of the study was a reduction of 40.4 ± 16.35 (range, 20.1 to 64.8) and 22.78 ± 9.11 (range, 9.1 to 44.7) in the percentage of
apoptotic CD4 and CD8 cells, respectively (P < .001 for both
subgroups). Representative cytofluorimetric profiles of these
experiments are shown in Fig 3. As expected in control individuals, the majority of CD4 and CD8 cells were not
stained by 7-AAD (Fig 3A). In contrast, HIV-positive individuals at
baseline (T0) had a very high frequency of CD4 and CD8 cells stained
with 7-AAD (ie, apoptotic cells) (Fig 3B); the L-carnitine treatment
(T4) strongly reduced the frequency of cells stained with 7-AAD to a
level comparable to that found in HIV-negative controls (Fig 3C).
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| Fig 1.
Percentage of CD4 cells undergoing apoptosis before and
after 15, 30, 90, and 150 days of L-carnitine treatment (each patient is indicated by a code). The apoptotic CD4 cells were analyzed by flow
cytometry after 7-AAD as described in Materials and Methods. ( )
2000; ( ) 5419; ( ) 5716; () 5762; ( ) 6438; () 6457; ( ) 6464; () 6466; ( ) 6655; () 6684; () 7173.
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| Fig 2.
Percentage of CD8 cells undergoing apoptosis before and
after L-carnitine treatment (each patient is indicated by a code). The
apoptotic CD8 cells were analyzed by flow cytometry after 7-AAD as
described in Materials and Methods. The symbols are the same as those
in Fig 1.
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| Fig 3.
Representative cytofluorographic assessment of CD4 and
CD8 cells stained with 7-AAD (apoptotic cells). Two-color
immunofluorescence staining was performed on CD4 and CD8 lymphocytes to
analyze the staining by 7-AAD. Representative results are shown for CD4
and CD8 T cells from a control individual (A) and an HIV-infected person at baseline (B) and after 150 days of L-carnitine treatment (C).
The vertical bar represents the threshold above which 7-AAD staining
was considered positive.
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The results of experiments performed by investigating the FSC/SSC
characteristics of CD4 and CD8 cells were comparable to the results
obtained by the 7-AAD staining method. In the representative experiments shown in Fig 4A, both CD4 and
CD8 cells in the HIV-positive donor (T0) shifted to a lower FSC/higher
SSC compartment, indicating that these lymphocytes were engaged into
the apoptotic pathway, a process that was not observed in lymphocytes
from the control individuals. The L-carnitine treatment (T4) of
HIV-positive patients resulted to shift both CD4 and CD8 cells to
higher FSC/lower SSC compartment, comparable to that observed in normal
individuals. Remarkably, CD4 and CD8 lymphocytes from the HIV-positive
donor (T0), which had decreased cell size (FSC), were precisely those that incorporated 7-AAD (Fig 4B). L-carnitine therapy (T4) led to an
increase of CD4 and CD8 cells with higher FSC, which did not
incorporate 7-AAD.
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| Fig 4.
Representative cytofluorographic experiment of apoptotic
CD4 and CD8 cells assessed by evaluating FSC and SSC parameters. Apoptotic cells are represented as a function of both parameters. (A)
Represents FSC/SSC lymphocyte compartments in an HIV-negative control
and an HIV-positive individual at baseline (T0) and after 150 days of
L-carnitine treatment (T4). A shift to a lower FSC/higher SSC was
observed in the HIV-positive individual compared with the normal
control, but L-carnitine treatment shifted FSC/SSC parameters to the
levels observed in the HIV-negative control. Accordingly, CD4 and CD8
cells in the HIV-positive individual at baseline (T0) with decreased
FSC were precisely those that incorporated 7-AAD (B). After 150 days of
L-carnitine treatment (T4), CD4 and CD8 cells with higher FSC did not
incorporate 7-AAD, as seen in the normal individual. The horizontal bar
represents the threshold above which 7-AAD staining was considered
positive.
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Ceramide levels.
The PBMC-associated ceramide levels were significantly higher at
baseline in the patient group in comparison to healthy individuals (10.2 ± 2.3 pmol/106 cells), according to our previous
work.8,20 L-carnitine therapy led to a progressive decline
in the ceramide levels with respect to the baseline (Fig 5
and Table 2). We measured decreased levels of PBMC-associated ceramide on each time point over the course of the
study and at the end of the study, the changes were statistically significant compared with the baseline (P = .003). The mean net change from baseline at the end of the study was a reduction of 35.8 ± 35.3 (range, decrease of 109 to a gain of 10)
pmol/106 cells. At each time point, the levels of ceramide
correlated only weakly to moderately with the frequency of apoptotic
CD4 and CD8 cells; a statistically significant correlation was found between ceramide levels and apoptotic CD4 cells on baseline and T1 (r = 0.71 and r = 0.73, P = .015 and .011, respectively)
(Table 3).
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| Fig 5.
Cell-associated ceramide levels expressed as
picomoles/106 cells before and after L-carnitine treatment
(each patient is indicated by a code). Fresh PBMC were isolated and the
lipid extracts were assayed for endogenous ceramide with the DAG-kinase
assay as described in Materials and Methods. The symbols are the same
as those in Fig 1.
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HIV-1 viremia.
HIV-1 viremia was measured at baseline, after 30 days, and at the end
of the study. The large variability in the baseline levels of plasma
HIV-1 RNA (log10) (range, 2.6 to 4.52 copies/µL) reflects
the differing viral burdens between patients (Table 1). Mean HIV
viremia levels increased slightly between baseline and T4 (mean
log10 gain = 0.22; median gain, 0.14; range, 0.45 to +1.09).
Safety of treatment.
No toxicity related to L-carnitine therapy was observed and none of the
subjects investigated required reductions in the dose of L-carnitine.
In addition, even though we did not observe any significant
improvements in the Karnofsky scores compared with the baseline (all of
the patients had a Karnofsky score of >90 at enrollment), all of the
subjects reported, with no exception, a sense of improved well-being by
the second week of L-carnitine treatment.
| |
DISCUSSION |
Recent work from our laboratory has shown that L-carnitine
downmodulates ceramide generation by interfering with the Fas-induced apoptotic signal,23 a major mechanism for the loss of CD4
and CD8 cells during the progression of the infection towards AIDS. The
finding that short-term treatment of AIDS patients with L-carnitine significantly reduced both the levels of lymphocyte-associated ceramide
and the frequency of apoptotic CD4 and CD8 cells22 added
weight to the in vitro results. Ceramide has been shown to initiate
signalling leading to lymphocyte apoptosis in response to several
mechanisms, which are implicated in the inappropriate apoptosis seen in
HIV-1-infected individuals, such as the chronic expression of Fas and
Fas ligand and the unregulated activation of the receptor for tumor
necrosis factor.13-17 Furthermore, ceramide enhances HIV-1
replication,18,19 and its cellular levels strongly increase
after experimental HIV-1 infection30 and in peripheral blood lymphocytes from HIV-infected patients.8 The role of ceramide in the progression of HIV-1 infection is also supported by the
finding that long-term nonprogressors, who remain clinically healthy
and immunocompetent over an extended period of time, have lower
lymphocyte-associated ceramide levels than subjects with AIDS.8,20
In this pilot study, we show evidence that L-carnitine therapy of
HIV-1-infected subjects may have a significant impact on CD4 absolute
counts, one independent predictor of the risk of developing
AIDS-related complications. We were unable to set up an appropriate
control group because we consider the placebo administration in AIDS
patients unethical. Moreover, in the same group of
patients, multiple CD4 counts were done before the initiation of the
study, therefore, excluding artifactual increases of the data generated during the trial period. The absolute CD4 cell counts increased over
the period when L-carnitine was administered and there was a
significant increase compared with the baseline after 90 and 150 days
of treatment, but a positive trend was already recognized after 15 and
30 days, even though the differences were not statistically significant.
The analyzed individuals represent a unique population of infected
subjects, all of whom were living in the Community of San Patrignano,
being exposed to the same environmental influences and with comparable
nutritional regimens. Remarkably, in the majority of individuals, there
was a past history of HBV or HCV infection and no opportunistic
infection was detected during the trial period.
They had stable CD4 counts over an extended period of time, but
progressively declining CD4 counts were recognized over the last 12 months of their stay in the Community before the enrollment in the
trial. Because they refused to undergo any treatment with antiretroviral drugs, an option for enrollment in the study was offered, even though none of them had carnitine deficiency as shown by
comparable plasma levels in healthy individuals.
The impact of L-carnitine therapy was not limited to CD4 cells, as even
CD8 cell absolute counts and rate of change exhibited a trend towards
improvement. Indeed, L-carnitine therapy resulted in an overall gain of
CD8 cells.
These results were paralleled by a strong reduction in the frequency of
apoptotic CD4 and CD8 cells and, even though this reduction occurred
gradually, changes in actual values between each time point and
baseline were always strongly significant.
The improvement in CD4 and CD8 absolute counts and the reduction in
apoptosis appear to have progressive decline in cell-associated ceramide levels as the common denominator. Furthermore, at each time
point, we found that ceramide levels correlated weakly to moderately
with the frequency of apoptotic CD4 and CD8 cells and the correlation
was statistically significant at baseline and T1 (r = 0.71 and r = 0.73, P = .015 and P = .011, respectively).
Remarkably, most of the above modifications occurred with only moderate
(± 0.5 log maximum) changes in the plasma viral load, with the
exception of one patient, in whom HIV-viremia increased by 1.1 log, but
the same individuals showed a significant increase in CD4 cell counts
and subjectively reported an increased sense of well-being and
resistance to fatigue. Taken together, our data suggest that long-term
L-carnitine administration may have a substantial impact on the chief
immunologic abnormality associated with HIV-1 infection, the loss of
CD4 T cells, through downmodulating the generation of ceramide and
reducing the rate of apoptotic lymphocyte death, without affecting the
HIV-1 viremia levels, thus suggesting that a dissociation exists
between changes in viremia and CD4 depletion. Indeed, L-carnitine
administration, although not affecting the viral life cycle, interrupts
the HIV-infection-associated ceramide generation and consequently the
lymphocyte apoptosis, which is considered a crucial factor in the
pathogenesis of AIDS.
Additional mechanisms contributing to the antiapoptotic effects of
L-carnitine, beside the inhibition of ceramide generation, cannot be
ruled out, as shown by recent studies reporting its ability to prevent
the disruption of mitochondrial transmembrane potential,31,32 an early and irreversible step in the
effector phase of apoptotic cell death.33 Moreover, an
abnormal redox cellular state is associated with HIV infection and
could be at least partially involved in the T-cell depletion in
AIDS.34,35 Indeed, oxidative stress has been implicated in
apoptosis, and it may provide effector mechanisms for the final common
pathway of programmed cell death. L-carnitine, when administered in
vivo, is very effective in inhibiting oxygen radical
production.36 Thus, the antiapoptotic effect of L-carnitine
could also be due to its antidepolarizing action at the mitochondrial
level and antioxidant activity.
The safety of treatment and the occurrence of toxicity related to the
treatment was closely monitored in our study. None of the subjects
investigated reported any toxicity related to L-carnitine therapy and
dose reductions were not necessary. As L-carnitine therapy was able to
increase CD4 counts in this small group of patients who had been
experiencing decline in CD4 counts, this suggests that such therapeutic
benefits may be real. The use of L-carnitine as an adjuvant to current
combination antiretroviral therapy in a randomized comparison against
the same therapies excluding L-carnitine is actually under
investigation.
In conclusion, our results indicate that L-carnitine targets the immune
system and suggest that complementing antiretroviral therapy with
L-carnitine may be an attractive approach to the management of
HIV-1-infected patients and a comparative evaluation seems justified.
The potential of L-carnitine to protect the host against the toxicity
of nucleoside analogs adds further weight to this
idea.37-40
| |
FOOTNOTES |
Submitted June 11, 1997;
accepted December 21, 1997.
Address reprint requests to Claudio De Simone, MD, PhD, Department of
Experimental Medicine, Via Vetoio 10, Coppito 2, 67100 L'Aquila,
Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract