IgG Antibodies in Patients with Pemphigus Vulgaris before and after Diagnosing with Immunofluorescence
Pemphigus is defined as a group of chronic self-immune vesicular diseases histologically recognized by inter-epidermic vesicles resulting from acantholysis. The aim of this study was to evaluate the precipitant and circulative IgG antibodies in patients with pemphigus vulgaris before and after treating with immunofluorescence. Sixty-two patients (34 females and 28 males) with clinically and pathologically confirmed P.V. were studied prospectively over a one year period of time during which direct and indirect immunofluorescent tests were performed before and after treatment. They had mild or moderate forms of disease. All patients received prednisolon 1-2 mg/kg/day and Azathioprine 2-3 mg/kg/day or methylpredisolon (1 g day-1 for 4 days) and cyclophosphamide (500 mg/first day) pulse therapy due to general condition. Thirty- four females and 28 males enrolled, the mean age were 39.5 years (SD = 12.7). Before treatment, 10 and 52 cases were positive for skin depositing +or++) and circulatory IgG (1/20 -1/60), respectively. Two to 3 month later, 37 were IgG positive with titers 1/20 to 1/160. The correlation between circulatory IgG before and after treatment was weakly positive (p = 0.05, r = 0.415). In the present study, treatment methods used for patients suffering from pemphigus vulgaris were not successful in significantly decreasing the circulative autoantibodies levels.
Received: December 08, 2012;
Accepted: February 16, 2013;
Published: March 28, 2013
The term of Pemphigus refers to a group of chronic fatal diseases
manifesting with mucocutaneous blisters. In pemphigus vulgaris which is the
most prevalent form of the disease, autoantibody is created against desmoglein
1 and 3 (Flores et al., 2012; Funakoshi
et al., 2012). The fatal disease usually appears in middle-aged persons
and rarely in youths with clinical, pathological and immunological symptoms
respectively as mucocutaneous blisters; acantholysis and blister formation between
cells of epidermis and complement and IgG precipitation in skin and circulative
antibodies (Goldust et al., 2011). Sometimes,
it is a sign of inner organs malignancies such as lung, digestive and reproductive
systems (Hatano et al., 2012). Classically,
the disease is treated with systemic glucocorticoids along with other immunosuppressives
including azathioprine. The main goal of treatment is to eradicate or decrease
pathogenic antibodies (Brandt et al., 2012;
Goldust et al., 2012; Ohyama
et al., 2012). The relation of these autoantibodies levels with severity
of the disease clinical symptoms is not exactly known. Some studies demonstrate
that serum level of circulative autoantibodies against keratinocytes determined
through indirect immunofluorescence method is in correspondence with severity
of disease activity (Dworschak et al., 2012;
Lotti et al., 2012). While, results obtained
from other studies do not indicate this direct relationship between clinical
symptoms and antibody levels (Behzad et al., 2012;
Hosoda et al., 2012).
Direct immunofluorescence is a standard method to trace precipitant IgG in
skin. The present study aims at determining precipitant and circulative IgG
antibodies before treatment in patients suffering from pemphigus vulgaris and
evaluating the effects of immunosuppressive drugs on levels of circulative antibodies
MATERIALS AND METHODS
In this cross sectional study of before and after intervention kind, 62 patients
with clinical and pathological symptoms of pemphigus vulgaris were studied for
one year from July 2011-2012. Written consent was obtained from all the patients.
This study was approved by ethic committee of Tabriz university of medical sciences.
Before treatment, IgG precipitation along with C3 in skin was traced
using direct immunofluorescence (DIF) and titer of IgG levels found in blood
circulation using indirect immunofluorescence (IIF) methods. After taking blood
samples from the patients, blood serum was isolated, to conduct IIF and added
to skin sections resulted from neonates circumcising which were fixed on the
slide so that it can connect to desmosomes found among keratinocytes in case
of existing of the circulative IgG antibodies. Then, human anti-IgG antibody
prepared from rabbits and marked with fluorescent materials were added and finally
studied under microscope. To do DIF, the sample resulted from biopsy of the
skin located around the lesions at the special immunology laboratory of Imam
Reza hospital, Tabriz was sectioned at micron sizes (using cryosection) and
was studied under fluorescent microscope using fluorescent conjugation after
coating to slide surface and its fixation. Depending on general conditions such
as age, records of hypertension and function of the interior organs, the patients
were classically treated with prednisolone (1-2 mg kg-1), azathioprine
(2-3 mg kg-1) or underwent pulse treatment (using methylprednisolone
1 g day-1 for 4 days and cyclophosphamide 500 mg day-1).
After relative recovery and decreasing at least 50% of the clinical symptoms
(lasting about 2-3 months) IIF of these patients were reexamined. Changing levels
of antibodies found in blood circulation is regarded as a criterion indicating
effectiveness of the applied treatment methods. The patients were studied considering
variables such as age, gender, disease duration, existence and severity of lesions
on skin, mouth and head before and after treatment. Considering disease severity,
the patients were divided into four groups: 1) non-morbidity of the desired
locus (skin, mouth and head, 2) slight form with morbidity of less than 1/3
of the desired place, 3) mild form with morbidity of 1/3-2/3 of the desired
place and 4) severe form with morbidity of more than 1/3 of the desired locus
(Table 1). The resulted outcomes were analyzed using SPSS-16
software and stated as mean±SD deviation. The results were regarded meaningful
if p>0.05 and highly correlate if r>0.5.
Mean age of these patients was 39.55±12.7 years. The youngest patient
was 20 and the oldest one was 79 years old. Mean of the disease starting period
was 1.93±1.03 years. The patients was consisted of 28 males (45%) and
34 females (55%). Considering skin morbidity, there were 36 cases (58.3%) of
skin non-morbidity and 26 patients (41.7%) of slight, mild, or severe morbidity
(Table 1). Considering oral morbidity, 32 cases (51.6%) of
oral non-morbidity and 30 cases of oral morbidity with different severities
were observed. Regarding head morbidity, there were 45 cases (73.3%) of non-morbidity
and only 17 cases of head morbidity as slight, mild, or severe (Table
1). Outcomes resulted from DIF on IgG precipitation in skin before starting
treatment demonstrated that DIF skin non-morbidity. In patients with skin lesions,
negative DIF were seen in 6 cases (23.7%), IgG+ precipitation in
9 patients (34.6%) and IgG++ precipitation in 11 cases (42.3%). Considering
IIF before treatment, it was negative in 10 patients (16.1%) but circulative
antibody was observed in 52 cases (83.9%) and its assay varied from 1/20 to
1/160 (Table 2). IIF tests after treatment demonstrated that
circulative antibody was not observed in 16 patients (30.2%) but it was seen
in 37 cases (69.8%). Repeating IIF test was not possible in 9 patients due to
their non-reference to the center. There were no cases of mortality in patients
during conducting the study. Evaluating correlation between circulative IgG
titer before starting the treatment with its titer after treatment revealed
that p = 0.005 and r = 0.415. Significant difference was not observed considering
mean of the obtained results on circulative IgG levels before and after treatment
(p = 0.122, r = 1.577).
There is no standard treating method and different methods are used to treat
and control the disease. The final goal is to eradicate or removing circulative
antibodies (Yokoyama et al., 2011) the disease
severity is controlled by clinical symptoms at acute stages. But, when the new
blisters do not appear and the previous lesions recover, changes of circulative
antibody levels will be helpful in determining drug dosage (Oberkirchner
et al., 2011).
||Prevalence and severity of clinical symptoms in patients suffering
from pemphigus vulgaris
|Values in brackets one percentage
||Levels of circulative IgG antibodies using IIF method before
and after treatment in patients suffering from pemphigus vulgaris
|Values in brackets one percentage
In the present study, there was no significant difference between mean of circulative
antibody before and after treatment (p = 0.122, t = 1.577). Correlation between
circulative antibody levels before and after treatment was positive but lower
than moderate level (p = 0.005, r = 0.415). The results demonstrate that decreasing
the amount of antibody after treatment was not valuable and the applied treatment
methods, in spite of improving the clinical symptoms, were not successful in
significantly decreasing the antibody levels. Several studies have been conducted
on how titer of these antibodies changes during treatment and different results
have been obtained (Lanza et al. 2010) according
to the reports, significant decrease of circulative antibodies levels after
successfully treating the disease using immunofluorescence test have been observed
in some studies (Prado et al., 2011). In a previous
study conducted on effects of plasmapheresis in a patient resisting against
prednisolone and cyclophosphamide, significant decrease of circulative antibodies
in immunofluorescence test associated with recovery of the clinical symptoms
was observed about 6 weeks after starting the treatment (Jennings
et al., 2011). In another study conducted on effects of intravenous
immunoglobulin (IVIg) in 21 patients suffering from severe form of pemphigus
vulgaris, significant decrease of serum antibodies levels have been reported
4-6 months after starting the treatment in indirect immunofluorescence (Wada
et al., 2011). But, other studies have presented different and, sometimes,
contrary results. In a study on effects of oral prednisolone in 46 patients
suffering from pemphigus vulgaris, decrease of circulative antibodies titer
after treating with immunofluorescence method has only been observed in patients
with severe disease. In patients with slight or moderate forms of the disease,
changes of the circulative antibodies levels was not considerable (Lanza
et al., 2011). In a previous study, effects of replacing plasma and
corticosteroid was evaluated in patients suffering from pemphigus vulgaris and
decrease of circulative antibodies levels in immunofluorescence was observed
in some patients. But, no change was seen in some other patients or it has been
increased in spite of improving of the clinical symptoms (Mejri
et al., 2011). Another study evaluated 78 patients suffering from
pemphigus vulgaris. In this study, circulative antibodies levels were decreased
in some patients after treatment using immunofluorescence method. But, no change
or even increasing Ab titer was observed in some other understudy patients.
In this study, evaluating response to treatment through measuring serial of
circulative antibodies levels is not recommended (Muller
et al., 2010). As observed, the above studies emphasize unpredictability
of changes of circulative antibodies levels in patients suffering from pemphigus
vulgaris. In the present study, mean of circulative antibodies levels were not
significantly changes (p = 0.1222, t = 1.5777) which may have different reasons.
Firstly, type of the applied treatment method is effective in changing circulative
antibodies levels (Endo et al., 2010). Simultaneous
use of several treatment methods results in more decrease of antibodies levels.
Secondly, changes of antibodies levels vary depending on the disease severity
(Santiago-et-Sanchez-Mateos et al., 2011). In
the present study, the patients suffered from slight and mild forms of the disease
and no case of the severe form was observed. The applied treatment methods were
not successful in significantly changing antibody levels because the patients
received different forms and dosages of corticosteroids prescribed by other
physicians in the outpatient ward before hospitalization. On the other hand,
the changes observed at circulative antibody levels vary depending on involvement
locus of the patients. Like the present study, mouth and head morbidity lead
to more stability of antibodies due to having high levels of antigens. Some
studies suggested that although special autoantibodies of pemphigus vulgaris
are of IgG kind, these antibodies have other subgroups (such as 1,3 kinds) and
only some of them are effective in pathogenesis process and common measuring
methods do not determine these differences (Aoyama et
al., 2010). On the other hand, methods used to measure antibodies levels
have different sensitivities, for example, ELISA method is more sensitive than
IIF. Additionally, dilution of the prepared samples will lead to different results.
In the present study, treatment methods used for patients suffering from pemphigus vulgaris were not successful in significantly decreasing the circulative autoantibodies levels. It is recommended that future studies pay more attention to kind and duration of the applied treatment methods, kind of skin, mucous, or oral morbidity and use of more sensitive evaluation methods such as Elisa to measure circulative antibodies.
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