Alpha 1 Acid Glycoprotein: Increased Serum and
Localized mRNA Expression as a Monitor for
Reactions in Leprosy
Astha Nigam, Itu Singh, Ravindra P. Turankar, Mallika Lavania and Utpal Sengupta
Stanley Browne Laboratory, The Leprosy Mission Trust India, New Delhi, India
Utpal Sengupta, Stanley Browne Laboratory, The Leprosy Mission Trust India, New Delhi, India. Pin-110093, Tel: +91-112-259-4295; E-mail:
Received: May 01, 2015; Accepted: July 13, 2015; Published: August 17, 2015
Citation: Nigam A, Singh I, Turankar RP, Lavania M, Sengupta U (2015) Alpha 1 Acid Glycoprotein: Increased Serum and Localized
mRNA Expression as a Monitor for Reactions in Leprosy. SOJ Microbiol Infect Dis 3(3): 1-4.
Alpha 1 Acid Glycoprotein (AGP) has been identified as a potentially
useful marker of clinical outcome in disease having inflammatory
component. The objective of this study is to determine the
levels of AGP with the possibility of using as a biomarker for Type 1
reactions (T1R) and Type 2 reactions (T2R) in leprosy. Serum levels
of AGP are evaluated in a total of 88 leprosy subjects [T1R; n = 32,
T2R; n =17 and Non-Reactional (NR); n = 39] using an Enzyme Linked
Immunosorbent Assay (ELISA). Relative quantification of mRNA encoding
AGP is also done from skin biopsies of same patients using
Real time PCR. The results revealed that the levels of serum AGP are
significantly higher in all leprosy cases with T1R and T2R when compared
to NR leprosy cases across the study groups (p < 0.05). The
Cross sectional analysis also revealed that the levels of mRNA expression
of AGP are significantly higher (p < 0.05) in leprosy cases with
both reactions when compared to non-reaction leprosy Cases (p <
0.05). Comparisons are made using Kruskal Wallis non parametric
Test. Significantly higher levels of AGP in serum and in the lesions of
patients may indicate its role as a predictive biomarker for type 1 and
2 reactions in leprosy.
Keywords: Alpha 1 Acid Glycoprotein; Biomarker; Leprosy;
Type 1 Reaction; Type 2 Reaction
Acute inflammation is one of the most essential host
responses to tissue injury or infection for the defense and
eventual restoration of tissue structure and function. Prolonged
inflammation, however, can contribute to the pathogenesis of
many diseases leading to loss of the function of tissue or organ
which often occurs in leprosy. Leprosy is a chronic infectious
disease caused by Mycobacterium leprae (M. leprae). M. leprae
mainly resides in macrophages of skin and Schwann cells of
peripheral nerves leading to neuropathy and loss of sensation
in the extremities of the body and in the area involved in the
skin. It is a spectral disease ranging from tuberculoid pole to
lepromatous pole with intermediate borderline forms.
Individuals with borderline disease Borderline Tuberculoid
(BT), borderline borderline/mid borderline (BB) and Borderline
Lepromatous (BL) experience T1R and T2R. T1R is an immunologically mediated episode that is a major cause of nerve
function impairment. Nerve function impairment may result in
disability and deformity . T2R occurs in Multibacillary (MB)
patients (Lepromatous leprosy (LL) and BL) . T2R mainly
occurs because of deposition of M. leprae antigen-antibody
complexes in tissues, which leads to complement activation
and neutrophil infiltration resulting in systemic inflammatory
response. Both the reactions are mainly responsible for nerve
damage that causes permanent disability in leprosy patients.
AGP, also called orosomucoid, are acute-phase protein that
can increase in plasma as much as 5-fold  in inflammation
during acute and chronic infections.AGP is a 43kDa protein that is
highly glycosylated . Due to the presence of sialic acids, AGP is
very negatively charged, its pi being only 2.7 . AGP is produced
mainly in the liver  although some extra-hepatic synthesis of
this protein has also been reported [7, 8]. The basal level of AGP
in serum is maintained at approximately 20 μmol/L in healthy
individuals. During an acute phase condition, the concentration
rises 2–5 times, making it one of the predominant proteins in
serum. Although AGP is an abundant protein, its real physiological
significance is not yet fully understood. Protective effects of AGP
against TNF-α induced pathogenesis have been described In vivo
 and gives non-specific resistance against lethal gram negative
infection . Keeping in mind both anti-inflammatory as well
as pro-inflammatory property of AGP [11,12] we designed the
present study. In the past, there has been one study indicated
high level of AGP in T2R  and showed its association with T2R
as a biomarker and an indicator of cure during treatment.
It is important that if manifestations of reactions in leprosy are
recognized early, then it would be possible for early intervention
with protective medicine like steroids and save the nerve and
tissue from the damage .We, therefore in this study, attempt
to examine the role of this protein in the development of both
type 1 and 2 reactions in leprosy.
Eighty eight newly diagnosed untreated leprosy patients with no reaction (NR) (n = 39) and with reactions (n =49; T1R =
32, T2R = 17) (ages between 10 and 60 years) were recruited
at TLM Community Hospital, New Delhi, India. All cases were
clinically examined by experienced dermatologists and were
classified on the histological scale of Ridley-Jopling (RJ)  from
ELISA for AGP measurement: Serum was separated from
5ml of venous blood followed by addition of protease inhibitor
cocktail (Sigma Aldrich Inc.) and stored at −200C until further
processing. Levels of AGP in serum samples were detected in
triplicate employing commercial ELISA kit (R&D System Inc.
USA) following the manufacturer's instructions.
RNA extraction, cDNA synthesis and quantitative PCR:
5×5 mm incisional skin biopsies taken from the edge of the
lesions were collected in RNA later for RNA extraction by using
TRI-reagent (Sigma–Aldrich) according to the manufacturer's
instructions. The concentration of RNA samples were determined
spectrophotometrically at 260/280nm. cDNA was constructed
from 1 μg of total RNA from each sample using Protoscript®
M-MuLV First Strand cDNA Synthesis Kit (New England Biolabs
Inc). Briefly, 1 μg of total RNA was mixed with Random Primer
mix and nuclease free water. RNA was then denatured at 700 C for
5 min. This is followed by the addition of Reaction Mix containing
buffer, Magnesium ions, dNTPs and Enzyme Mix containing
0.5 units/μl of Reverse Transcriptase and RNase Inhibitor.
Temperature cycling conditions included 25° C for 5 min, 45° C
for 1 h followed by inactivation of enzyme at 80°C for 5 min.
Two microliters of cDNA was used per 20 μl reaction. The
reaction was carried out using the SYBR Green PCR Master Mix
on Rotor Gene Q (Qiagen Inc. USA) using gene specific primer
set designed for this study (Table 1). Each experiment was
performed in duplicate.
The statistical analysis was performed using Graph-Pad
Prism software (Version 6). ANOVA was used to compare
means of clinical laboratory parameters. The Kruskal-Wallis
nonparametric test was used to test differences between
responses among three groups and post test (Dunn's test) was
applied to find out the significant difference between NR and
T1R/T2R group. All statistical analyses were two sided, and a p
value of < 0.05 was considered statistically significant. Graphs are
shown as mean ± Standard Error of Mean (SEM) by using Graph
pad prism, two sided table, n-1 degree of freedom, alpha=0.05,
95% Confidence Interval.
The real-time data were analyzed on Rotor-Gene Q Series
Software (Software Version 2.0.2). An association with p value <
0.05 was considered as statistically significant.
Serum levels of AGP across the study groups
Analysis of AGP across the study groups reveal that the levels
are significantly higher in leprosy cases with T1R and T2R when
compared to that of NR group (T1R Vs NR mean± SEM= 464502 ±
26333 vs 231485 ± 22843, T2R Vs NR 450255 ± 41659vs231485
± 22843, p<0.05 (Figure 1)
Analysis of mRNA expression profiles of GAPDH and
AGP in the skin lesions
We analyzed mRNA expression profile by calculating fold
difference in expression using Pfaffl method (16) based on the
formula mentioned below:
The percentage efficiency of the primers from the standard
graphs was determined to be in the order of 98% for GAPDH
(Glyceraldehyde 3 Phosphate Dehydrogenase) (House Keeping
Gene) and 97% for AGP.
Individual expression ratio using the above formula was
calculated for cases in T1R, T2R and NR leprosy taking the
average ratio of NR as the control value for all the cases.
Coincident measurement of GAPDH gene has been used for the
normalization of target gene expression data.
Cross sectional analysis of mRNA expression levels of AGP
across the study groups revealed that the levels are significantly
higher in leprosy cases with T1R and T2R when compared to NR
group (T1R Vs NR and T2R Vs NR, mean mRNA Expression Ratios
± SEM = 36.45 ± 19.61 Vs11.02 ± 4.817 and 64.49 ± 17.60 Vs
11.02 ± 4.817, p<0.001). Although no significant difference was
found between T1R and T2R (Figure 2).
In an attempt to identify a biomarker for detection of the
onset of reactions in leprosy we analyzed mRNA expression
profile and serum levels of AGP in leprosy patients with type 1
and type 2 reactions along with a control group of patients having
no reactions at the time of recruitment. We have identified a
statistically significantly high level of AGP in serum of both types
of leprosy reactions. The higher AGP levels as reflected in the
serum profile, in active reaction patients than in controls with
no reaction could be an early indication of disease progression.
AGP is an inflammation related protein. The several fold increase
of AGP concentration in the circulation during an acute phase
Table 1: Primer set for mRNA expression.
Length of Amplicon
GAPDH (Housekeeping gene)
(in house designed primers)
Figure 1: Levels of AGP in serum samples of NR (n =3 9), T1R (n = 32)
and T2R (n = 17) of leprosy patients using ELISA. (*** p < 0.0001)
Figure 2: mRNA expression ratios of AGP/GAPDH in lesional skin biopsies
of NR (n =39), T1R (n =32) and T2R (n =17) leprosy patients using
real time PCR. (** p < 0.001)
response could influence the biological functions of the molecule
in humans . Although the detailed biological functions of AGP
have not been elucidated completely, the major physiological
roles of AGP reported so far involve the binding and transport
of a range of drugs and it has immunomodulating effects as
well . This protein induces monocytes and macrophages to
synthesis pro-inflammatory cytokines IL-6, IL-12 and expression
of molecules that inhibit the activity of IL-1β and TNF-α by IL-1
receptor antagonist and soluble TNF receptor . This cytokinepattern
suggests that AGP might have a dual effect; inducing
both a pro-inflammatory environment and an anti-inflammatory
environment, depending on the circumstances and the phase of
the disease . Leprosy presents a spectrum of immunological
groups between the two poles, the tuberculoid pole (TT) with
increased cell mediated immunity, which gradients towards
the lepromatous pole (LL) through a series of borderline forms
(BT, BB and BL) with simultaneous increase in humoral immune
response and increase in the bacillary load. Type1 reaction
(T1R) also called Reversal Reaction (RR), particularly occurs
in BT through BL types of leprosy with exacerbations of the
pre-existing skin and nerve lesions due to up-gradation of Cell
Mediated Immunity (CMI) more often during and after Multi
Drug Therapy (MDT) . Dynamics of immunological reactions could reflect by changes in the levels of certain circulatory
molecules that could act as possible biomarkers for predicting
reactions in leprosy (20). A Predictive biomarker for prediction
of inflammatory reactions in leprosy has long been desired. In
the present study, we have evaluated the diagnostic efficacy of
the AGP in T1R and T2R of leprosy that lead to nerve function
impairments in the patients. The present study focused on the
presence of AGP in circulation and lesions in reactional state
of leprosy. We here demonstrated, using ELISA and Real-Time
PCR analysis the presence of AGP, in serum and skin from
active lesions of patients with leprosy reactions including skin
smear negative BL and BT leprosy patients. Not only systemic
but localized levels of AGP measured in terms of relative gene
expression revealed that this acute phase protein coding gene
is over expressed in lesional skin mRNA which indicates its
active role in regulation of inflammatory episodes at the lesional
levels. Although,no significant difference in AGP production
was observed between T1R and T2R group. But when taken
together, these data suggest that AGP levels correlate with
reactional state in leprosy. However, this study has limitations
inherent to its cross-sectional design that cannot determine a
causal relationship. It may also be assumed that our sample size
is relatively small. More evidence is required to evaluate the
association between high levels of AGP, and reactional state of
leprosy. Since this study should be considered as preliminary,
the consistency of the association might be explored in other
clinical studies monitoring the common inflammatory mediators
(CRP, IL-6, TNF-α etc.), including AGP, in leprosy patients with
T1R, T2R and in patients without any reaction as controls. The
present study has several strengths. It is the first study to deal
with the association between clinical parameters and T1R, T2R
in a leprosy population. We chose untreated leprosy patients
with T1R and T2R as a model because it represents the exact
role of AGP, in developing the inflammatory condition. To
our knowledge, this is the first clinical study demonstrating a
systemic link between AGP and inflammation in leprosy using
real-time expression for localized along with serum circulatory
levels. A prospective cohort study involving measurement of AGP
levels, before, during and after the occurrence of T1R, T2R and
during MDT treatment may provide predictive information on
the effectiveness of AGP as a biomarker to identify patients going
to manifest reactions in leprosy.
Acute-phase proteins produced by cytokine activity are useful
diagnostic markers that could also be used to monitor treatment
response as they can be serially quantified and used as regression
markers of the inflammatory response during treatment the
quantification of proteins during the course of various acute and
chronic inflammatory disorders is useful in diagnosis, therapy,
and in some cases, prognosis. The AGP is measurable biomarker
that correlates reactions in leprosy and may be indicative factor
of conditions favorable to nerve damage, as it shows a significant
increase in type 1and 2 reactions both in terms of levels in serum
and also in their mRNA expression. The conclusions of our study
support the hypothesis that localized, persistent infection may
influence systemic levels of inflammatory mediators. However, larger prospective studies are needed to establish its utility as
a biomarker that may be predictive factor of clinical outcome in
This study was part of Project ID No: 2010-12950 granted
from the "Indian Council of Medical Research-New Delhi". We
extend our special thanks to all the participants who volunteered
for the study. We would like to thank The Leprosy Mission (TLM)
Trust India, the host organization, and the entire associated
medical staff. The authors declare that they have no conflict of
interest. The authors alone are responsible for the content and
writing of the paper. This study was approved by the Ethical
Committee of The Leprosy Mission Trust India (TLMTI) and
written informed consent was obtained from all patients and
- Stefani MM, Guerra JG, Sousa AL, Costa MB, Oliver ML, Martelli CT, et
al. Potential plasma markers of Type 1 and Type 2 leprosy reactions:
a preliminary report. BMC Infect Dis. 2009; 9:75. doi: 10.1186/1471-
- Kahawita IP, Walker SL, Lockwood DNJ. Leprosy type 1 reactions and
erythema nodosum leprosum. An Bras Dermatol. 2008;83(1):75-82.
- Kremer JM, Wilting J, Janssen LH. Drug binding to human alpha-1-acid
glycoprotein in health and disease. Pharmacol Rev. 1988; 40(1):1-47.
- Kushner I. The acute phase response: an overview. In: Mackiewicz,
A, Kushner I, Baumann H, editors. Acute phase proteins: molecular
biology, biochemistry, and clinical applications. Boca Raton: CRC
Press;1993. p. 3–19.
- Schmid K. Alpha 1-Acid glycoprotein. In: Putman FW, editor. The
plasma proteins: structure function and genetic control. New York:
- Gahmberg CG, Andersson LC. Leukocyte surface origin of human
alpha1-acid glycoprotein (orosomucoid). J Exp Med. 1978;
- Eap CB, Baumann P, Moretta A. Synthesis of Alpha 1-acid glycoprotein
by human T lymphocytes. Experientia. 1989;45:A 52.
- Sörensson J, Matejka GL, Ohlson M, Haraldsson B. Human endothelial
cells produce orosomucoid, an important component of the capillary
barrier. Am J Physiol. 1999;276(2 Pt 2):H530-4.
- Libert C, Brouckaert P, Fiers W. Protection by alpha 1-acid
glycoprotein against tumor necrosis factor-induced lethality. J. Exp.
- Hochepied T, Van Molle W, Berger FG, H Baumann, C Libert.
Involvement of the acute phase protein α1-acid glycoprotein in
nonspecific resistance to a lethal Gram-negative infection. J. Biol.
- Boutten A, Dehoux M, Deschenes M, Rouzeau JD, Bories PN, Durand
G. Alpha 1-Acid glycoprotein potentiates lipopolysaccharide-induced
secretion of interleukin-1beta, interleukin-6 and tumor necrosis
factor-alpha by human monocytes and alveolar and peritoneal
macrophages. Eur.J.Immunol. 1992;22(10):2687–95.
- Su SJ, Yang BC, Wang YS, Yeh TM. Alpha1-Acid glycoprotein-induced
tumor necrosis factor-a secretion of human monocytes is enhanced
by serum binding proteins and depends on protein tyrosine kinase
activation. Immunopharmacology. 1999;41(1):21-9.
- Gupta N1, Shankernarayan NP, Dharmalingam K.. Alpha 1-Acid
glycoprotein as a putative biomarker for monitoring the development
of the type II reactional stage of leprosy. J Med Microbiol. 2010;59(Pt
4):400-7. doi: 10.1099/jmm.0.016394-
- Lockwood DN, Suneetha S. Leprosy: too complex a disease for a simple
elimination paradigm. Bull World Health Organ. 2005;83(3):230-5.
- 15. Ridley DS, Jopling WH. Classification of leprosy according to
immunity: a five group system. Int J Lepr Other Mycobact Dis.
- Pfaffl MW. A new mathematical model for relative quantification in
real-time RT-PCR. Nucleic Acids Res. 2001;29(9):e45.
- Fournier T, Medjoubi N N, Porquet D. Alpha 1 acid glycoprotein.
Biochim. Biophys. Acta 2000;1482(1-2):157-71.
- Tilg H, Vannier E, Vachino G, Dinarello CA, Mier JW. Antiinflammatory
properties of hepatic acute phase proteins: preferential induction
of interleukin 1 (IL-1) receptor antagonist over IL-1 beta synthesis
by human peripheral blood mononuclear cells. J. Exp. Med.
- Hochepied T, Berger FG, Baumann H, Libert C. Alpha(1)-Acid
glycoprotein: an acute phase protein with inflammatory and
immunomodulating properties. Cytokine Growth Factor Rev.
- Scollard DM, Smith T, Bhoopat L, Theetranont C, Rangdaeng S, Morens
DM. Epidemiologic characteristics of leprosy reactions. Int J Lepr
Other Mycobact Dis. 1994;62(4):559-67.