500
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION
NOVEMBER, 1976
Electrophoretic Patterns of Serum Proteins and Immunoglobulin Levels in Mycobacterial Infections-Studies in Mice Infected with Mycobacterium Leprae and Mycobacterium Lepraemurium* R. G. NAVALKAR, P. J. PATEL,, R. R. DALVIt AND M. V. KANCHANA, Departnment of Microbiology,
Meliarry Medical College, Nashiville, Tenniiessee
STUDIES on evaluation of various serum proteins,`6 in addition to changes in serum Lactic dehydrogenase7 and antibodies to mycobacterial antigens8 have been carried out in the past number of years by several workers in regard to the various stages of leprosy infection. These studies have primarily been directed against patients suffering from the disease. In more recent years, with the establishment of the mouse as a model for M. leprae infections,9 it has become a vital tool for evaluating, not only the efficiency of various chemotherapeutic agents, but also for the evaluation of both the humoral and the cellular immune responses. The parameters used in these studies, such as the antibody forming cell,10 detection of delayed type hypersensitivity11 and the ability of the mouse lymphocytes to undergo blast transformation12 have led to the observation that the mouse model may, when infected with M. leprae fall within the category of the early stage of leprosy infection in the spectrum of human leprosy. On the other hand, similar studies conducted using mice infected with the rat leprosy bacillus (M. lepraemurium) have indicated that this disease represents the advanced stage of leprosy infection."3 Present studies were undertaken as an *Supported by the U.S. Leprosy Panel of the U.S.Japan Co-operative Medical Science Program administered by the Geographic Medicine Branch, National Institute of Allergy and Infectious Diseases. (Grant R22 AI-08647). tCurrent address: Trudeau Institute Inc., Saranac Lake, New York. tCurrent address: Tuskegee Institute, Alabama.
extension of the above mentioned studies, with a view to confirming the placement of the mouse model infected with M. leprae into the early stage and the mouse model infected with M. lepraemurium in the advanced stage of leprosy infection. Parameters used in these studies were, the assay of serum immunoglobulins, the estimation of serum proteins and changes therein, if any. As a comparative control similar assays were carried out on sera obtained from patients suffering from leprosy. MATERIALS AND METHODS
Mice. Six to eight weeks old female inbred strains of BALB/c mice, bred and raised in the laboratory of Dr. L. Levy, USPHS Hospital, San Francisco, were used throughout the experiments. Infection and Immunization. a) M. leprae. Five groups of mice were used. Each group consisted of 60 mice. Groups A and B were inoculated with 5 x 1 0 live and dead M. leprae bacilli respectively, in the right hind foot pad. Groups C and D received extracts of mouse foot pad material prepared from infected and uninfected mice respectively. Group E consisted of normal uninoculated mice. b) M. lepraemurium. Three groups of 60 mice each were studied. One group (Z) was infected with 109 viable bacilli of Hawaiian strain of Mycobacterium lepraemurium (Mlm) by intraperitoneal inoculation. A second group (Y) was given 109 nonviable Mlm bacilli intraperitoneally. The third group (X) received neither viable nor
Vol. 68, No. 6
Mycobacterial Infections
a non-viable challenge. For the non-viable challenge, the Mlm was killed by five repeated cycles of freeze thawing. Collection of Plasma. Four mice from each group for M. leprae and three from each group for M. lepraemurium were sacrificed at biweekly intervals beginning the week of infection and extending up to the 14th week post infection for various experiments. Prior to sacrifice, blood was collected by intra-orbital puncture, into sterile tubes containing herparin-blood from all the mice in each group pooled in order to obtain sufficient quantities of plasma for various other assays. Plasma was collected aseptically and stored at -20°C until used. Sera from leprosy patients. Sera from leprosy patients in various stages of infection viz lepromatous, (LL), tuberculoid (TT) and borderline (BB), and from contacts and healthy controls were obtained from The Acworth Leprosy Hospital, Bombay, India. The classification of the cases is in accordance with the data obtained from the A.L. Hospital. Serum protein determinations. Electrophoresis of each sample was carried out in a commercially obtained electrophoretic apparatus on thin agarose Pol-E films. (Pfizer Diagnostics, Pfizer Inc., Clifton, New Jersey). Samples were loaded in the wells using a microlitre pipet. On each film, one well was always filled with normal serum and the rest with test sera. Buffer used was also obtained from the same company. The buffer contained known concentration of sodium barbital, EDTA and 2N HCl and had a pH of 8.6+-0.2. Each run was at 90-95 volts for 40 minutes. The migrated Pol-E film was then stained with a 0.2% amido black lOB stain for 15 minutes, washed for 30 seconds in 5% acetic acid solution, dried in an oven at 72°C for 20 minutes and destained in three successive changes of 5% acetic acid for two minutes each. The film was then removed, allowed to dry for 15 minutes at 72°C and cut into strips for scanning by a scanning densitometer at 580 nm.
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JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION
502
Immunoglobulin assay. Commercially obtained (Meloy Laboratories) radial immuno diffusion plates, impregnated with monospecific mouse and human immunoglobulins respectively, were used for this assay. A standard reference curve was first drawn using respective antiglobulin sera (Meloy Laboratories) in order to facilitate evaluation of the results. Each plate was filled with the standard antiserum at various dilutions and incubated in moist atmosphere for 16 to 18 hours at room temperature. Diameter of the precipitin ring around each well was measured using the standard magnifier and recorded. The results were plotted on a semi-log graph paper and the best straight line was drawn between points. This served as a reference curve for the immunoglobulin assays of the test and control sera. RESULTS
The experiments were designed to study the changes in the serum proteins and immunoglobulins of mice infected and/or immunized with M. leprae and M. lepraemurium. Serum protein determinations. Table 1 gives the results of agarose electrophoresis done on sera of mice in the M. leprae groups. In all five groups the levels of albumin and alpha and beta globulins remained almost constant throughout the experiments while the gamma globulin showed an increase in animals of group A as the
NOVEMBER, 1976
infection progressed. However, the degree of increase was not as high as expected. Table 2 shows the serum protein levels of mice in all the three groups in the M. lepraemurium studies. As can be seen from this table, in the infected group there appears to be an initial increase in the prealbumin region which persists throughout the course of studies, whereas albumin and alpha and beta globulins appear to fluctuate within the groups. On the other hand there is a steady increase of gamma globulin once again at a level not as high as expected. The point of interest in both the studies is the presence of pre-albumin levels in mice that were infected or immunized, either with M. leprae or M. lepraemurium, regardless of the challenge being viable or non-viable. No such situation was noticed either in control animals or patients suffering from leprosy and their contacts. Results of the electrophoretic studies on sera from leprosy patients are presented in Table 3. A significant difference is noted between patients with lepromatous leprosy and tuberculoid leprosy in regard to the levels of gamma globulins. Increased gamma globulin levels were noticed in sera from lepromatous patients as against almost normal levels in the sera from tuberculoid patients. Patients in the borderline stage of the clinical spectrum and the contacts exhibited a slightly raised gamma globulin level. These results are in agreement with
Table 2. SERUM PROTEIN ANALYSIS-MICE INFECTED/IMMUNIZED WITH M. LEPRAEMURIUM
Proteins 2nd week 6th week 12th week 16th week
a
=
x,
60.6 3.9 54.7 18.4 4.0 56.0 20.3 0.2 62.8 22.0 2.1
Z
Y
X
Group*
c4 17.6 11.4 15.8 2.0
C
15.8 15.5 4.0 13.1
ac
6.3 51.0 26.1 14.3 2.3 53.2 23.0 5.1 5.5 75.9 18.6 56.5 24.2 4.4
*X = Uninfected normal mice Y = I.P. Challenge with 109 non-viable M. lepraemurium Z = I.P. Injection with 109 viable M. lepraemurium
2.4 16.3 16.9
6.2 2.2 5.9 1.9
46.5 13.3 14.8 19.1 46.5 22.0 5.6 25.5 39.6 11.2 22.9 20.6 51.8 5.0 11.5 29.4
503
Mycobacterial Infections
Vol. 68, No. 6
Table 3. SERUM PROTEIN ANALYSIS AND IMMUNOGLOBULIN ASSAY* -LEPROSY PATIENTS
Tuberculoid (11) Lepromatous (12) Borderline (5) Contacts (7) Healthy controls (4) *
=
Immunoglobulin
Serum Protein
Type
Prealb
Alb
A lpha
Beta
Gamma
IgA
IgG
-
59.98 53.8 59.94 70.07 60.17
20.05 15.2 15.10 10.24 17.65
14.0 10.73 17.94 11.44 17.55
5.78 18.62 6.5 8.07 4.62
290.0 546.0 314.0 ND
1577.1 2268.0 1628.0 ND 1000.0
-
220
IgM 156.0 279.6 206.4 ND 98.0
Values expressed in mg%
the results of the studies on humoral antibodies in patients.14 Immunoglobulin assays. These assays were carried out on sera from all groups of mice and those from patients. Table 4 gives the results of the immunoglobulin assay on M. leprae groups of mice. Compared to the normal values, the other groups show an increased level of all the classes of immunoglobulins viz., IgA, IgG1 and IgG2 and IgM. Among these IgA and IgG1 appear to increase as the infection progresses whereas the other two, IgG2 and IgM remain constant. Group A (infected) exhibits and increased level of IgG2 as well. Table 5 gives the data on serum immunoglobulins in mice infected or immunized with M. lepraemurium. The infected group (Group Z) shows elevated levels of all the classes of immunoglobulins compared to the other two groups. Among these IgA, and IgG1 seem to increase as the infection progresses while the other classes viz IgG2a, IgG.,b and IgM tend to remain constant. This is somewhat similar to the situation seen in mice infected with M. leprae. Immunoglobulin assays on sera from patients revealed that IgG increases as the clinical spectrum shifts from the early stages (TT) to advanced stages (LL), through borderline (BB), while the reverse is seen with IgA which decreases as the disease progresses. Increased IgM levels are noticed in sera from lepromatous patients as compared to those observed in sera from either the tuberculoid and borderline patients or from contacts of leprosy patients
and healthy individuals. These results are summarized in Table 3. DISCUSSION
Present studies were undertaken with a view to determining changes, in the serum proteins, and the levels of immunoglobulins of mice infected with M. leprae and M. lepraemurium and to compare them with those found in leprosy patients. Studies on serum proteins and immunoglobulins as well as the humoral and cell mediated immune response have been carried out by many investigators on patients in various stages of leprosy infection.5'8,'012'13'15 Since the time the mouse model has been recognized as a useful tool for the growth of M. leprae and for evaluation of anti-leprosy drugs, several investigations have been carried out on the immune response in mice infected with M. leprae.16-18 Previous work done in this laboratory on mice infected with M. leprae and M. lepraemurium on humoral as well as cell mediated immune response were found to be supportive of the view that the mouse model when infected with M. leprae could represent the early stage of human leprosy while the M. lepraemurium infection in mice could resemble an advanced stage of the disease. 10-13 Current studies were designed to see whether the serum protein changes and changes in the immunoglobulin levels also lend support to this contention. The agarase electrophoretic studies of serum protein analysis on sera from mice infected with
504
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION
M. leprae, showed that the levels of gamma globulin in these mice are slightly higher than the normal values and that there is an increase in the gamma globulin level as the disease progresses. Similar situation is observed in mice infected with M. lepraemurium but the levels of gamma globulin are comparatively higher. Serum protein analysis of sera from patients with leprosy indicated that in early phase of the disease the gamma globulin level remains close to that seen in uninfected individuals. However, in the advanced stages of infection, the levels are rather high. Borderline cases and contacts fall within these two extremes. The results clearly indicate that the gamma globulin levels of mice infected with M. leprae are significantly similar to those observed in sera from tuberculoid leprosy patients while those of M. lepraemurium infected mice resemble the levels of gamma globulin observed in sera from lepromatous leprosy patients. The data obtained in the immunoglobulin assays have revealed increased immunoglobulin levels in mice treated with M. leprae and M. lepraemurium. Among the three classes of immunoglobulins studied the IgM levels were constant while the IgG and 1LA were seen to increase as the disease progressed. Between the two subclasses of IgG, the IgG, levels were seen to increase while IgG., remained constant. Immunoglobulin assay on sera from patients indicated that the levels of the three classes of immunoglobulins were higher than that of healthy controls. While IgG seems to increase proportionately with the progression of the disease from an early stage to the advanced stage; the IgA appears to decline, whereas, the IgM levels remain constant. The observations made in the current studies indicate a trend towards the placement of the mouse model infected with M. leprae in the early stage of leprosy infection in humans, whereas that infected with M. lepraemurium fits within the spectrum of the advanced stages of the disease. The data tend to support our previous ob-
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NOVEMBER, 1976
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JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION
NOVEMBER, 1976
Electrophoretic Patterns of Serum Proteins and Immunoglobulin Levels in Mycobacterial Infections-Studies in Mice Infected with Mycobacterium Leprae and Mycobacterium Lepraemurium* R. G. NAVALKAR, P. J. PATEL,, R. R. DALVIt AND M. V. KANCHANA, Departnment of Microbiology,
Meliarry Medical College, Nashiville, Tenniiessee
STUDIES on evaluation of various serum proteins,`6 in addition to changes in serum Lactic dehydrogenase7 and antibodies to mycobacterial antigens8 have been carried out in the past number of years by several workers in regard to the various stages of leprosy infection. These studies have primarily been directed against patients suffering from the disease. In more recent years, with the establishment of the mouse as a model for M. leprae infections,9 it has become a vital tool for evaluating, not only the efficiency of various chemotherapeutic agents, but also for the evaluation of both the humoral and the cellular immune responses. The parameters used in these studies, such as the antibody forming cell,10 detection of delayed type hypersensitivity11 and the ability of the mouse lymphocytes to undergo blast transformation12 have led to the observation that the mouse model may, when infected with M. leprae fall within the category of the early stage of leprosy infection in the spectrum of human leprosy. On the other hand, similar studies conducted using mice infected with the rat leprosy bacillus (M. lepraemurium) have indicated that this disease represents the advanced stage of leprosy infection."3 Present studies were undertaken as an *Supported by the U.S. Leprosy Panel of the U.S.Japan Co-operative Medical Science Program administered by the Geographic Medicine Branch, National Institute of Allergy and Infectious Diseases. (Grant R22 AI-08647). tCurrent address: Trudeau Institute Inc., Saranac Lake, New York. tCurrent address: Tuskegee Institute, Alabama.
extension of the above mentioned studies, with a view to confirming the placement of the mouse model infected with M. leprae into the early stage and the mouse model infected with M. lepraemurium in the advanced stage of leprosy infection. Parameters used in these studies were, the assay of serum immunoglobulins, the estimation of serum proteins and changes therein, if any. As a comparative control similar assays were carried out on sera obtained from patients suffering from leprosy. MATERIALS AND METHODS
Mice. Six to eight weeks old female inbred strains of BALB/c mice, bred and raised in the laboratory of Dr. L. Levy, USPHS Hospital, San Francisco, were used throughout the experiments. Infection and Immunization. a) M. leprae. Five groups of mice were used. Each group consisted of 60 mice. Groups A and B were inoculated with 5 x 1 0 live and dead M. leprae bacilli respectively, in the right hind foot pad. Groups C and D received extracts of mouse foot pad material prepared from infected and uninfected mice respectively. Group E consisted of normal uninoculated mice. b) M. lepraemurium. Three groups of 60 mice each were studied. One group (Z) was infected with 109 viable bacilli of Hawaiian strain of Mycobacterium lepraemurium (Mlm) by intraperitoneal inoculation. A second group (Y) was given 109 nonviable Mlm bacilli intraperitoneally. The third group (X) received neither viable nor
Vol. 68, No. 6
Mycobacterial Infections
a non-viable challenge. For the non-viable challenge, the Mlm was killed by five repeated cycles of freeze thawing. Collection of Plasma. Four mice from each group for M. leprae and three from each group for M. lepraemurium were sacrificed at biweekly intervals beginning the week of infection and extending up to the 14th week post infection for various experiments. Prior to sacrifice, blood was collected by intra-orbital puncture, into sterile tubes containing herparin-blood from all the mice in each group pooled in order to obtain sufficient quantities of plasma for various other assays. Plasma was collected aseptically and stored at -20°C until used. Sera from leprosy patients. Sera from leprosy patients in various stages of infection viz lepromatous, (LL), tuberculoid (TT) and borderline (BB), and from contacts and healthy controls were obtained from The Acworth Leprosy Hospital, Bombay, India. The classification of the cases is in accordance with the data obtained from the A.L. Hospital. Serum protein determinations. Electrophoresis of each sample was carried out in a commercially obtained electrophoretic apparatus on thin agarose Pol-E films. (Pfizer Diagnostics, Pfizer Inc., Clifton, New Jersey). Samples were loaded in the wells using a microlitre pipet. On each film, one well was always filled with normal serum and the rest with test sera. Buffer used was also obtained from the same company. The buffer contained known concentration of sodium barbital, EDTA and 2N HCl and had a pH of 8.6+-0.2. Each run was at 90-95 volts for 40 minutes. The migrated Pol-E film was then stained with a 0.2% amido black lOB stain for 15 minutes, washed for 30 seconds in 5% acetic acid solution, dried in an oven at 72°C for 20 minutes and destained in three successive changes of 5% acetic acid for two minutes each. The film was then removed, allowed to dry for 15 minutes at 72°C and cut into strips for scanning by a scanning densitometer at 580 nm.
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JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION
502
Immunoglobulin assay. Commercially obtained (Meloy Laboratories) radial immuno diffusion plates, impregnated with monospecific mouse and human immunoglobulins respectively, were used for this assay. A standard reference curve was first drawn using respective antiglobulin sera (Meloy Laboratories) in order to facilitate evaluation of the results. Each plate was filled with the standard antiserum at various dilutions and incubated in moist atmosphere for 16 to 18 hours at room temperature. Diameter of the precipitin ring around each well was measured using the standard magnifier and recorded. The results were plotted on a semi-log graph paper and the best straight line was drawn between points. This served as a reference curve for the immunoglobulin assays of the test and control sera. RESULTS
The experiments were designed to study the changes in the serum proteins and immunoglobulins of mice infected and/or immunized with M. leprae and M. lepraemurium. Serum protein determinations. Table 1 gives the results of agarose electrophoresis done on sera of mice in the M. leprae groups. In all five groups the levels of albumin and alpha and beta globulins remained almost constant throughout the experiments while the gamma globulin showed an increase in animals of group A as the
NOVEMBER, 1976
infection progressed. However, the degree of increase was not as high as expected. Table 2 shows the serum protein levels of mice in all the three groups in the M. lepraemurium studies. As can be seen from this table, in the infected group there appears to be an initial increase in the prealbumin region which persists throughout the course of studies, whereas albumin and alpha and beta globulins appear to fluctuate within the groups. On the other hand there is a steady increase of gamma globulin once again at a level not as high as expected. The point of interest in both the studies is the presence of pre-albumin levels in mice that were infected or immunized, either with M. leprae or M. lepraemurium, regardless of the challenge being viable or non-viable. No such situation was noticed either in control animals or patients suffering from leprosy and their contacts. Results of the electrophoretic studies on sera from leprosy patients are presented in Table 3. A significant difference is noted between patients with lepromatous leprosy and tuberculoid leprosy in regard to the levels of gamma globulins. Increased gamma globulin levels were noticed in sera from lepromatous patients as against almost normal levels in the sera from tuberculoid patients. Patients in the borderline stage of the clinical spectrum and the contacts exhibited a slightly raised gamma globulin level. These results are in agreement with
Table 2. SERUM PROTEIN ANALYSIS-MICE INFECTED/IMMUNIZED WITH M. LEPRAEMURIUM
Proteins 2nd week 6th week 12th week 16th week
a
=
x,
60.6 3.9 54.7 18.4 4.0 56.0 20.3 0.2 62.8 22.0 2.1
Z
Y
X
Group*
c4 17.6 11.4 15.8 2.0
C
15.8 15.5 4.0 13.1
ac
6.3 51.0 26.1 14.3 2.3 53.2 23.0 5.1 5.5 75.9 18.6 56.5 24.2 4.4
*X = Uninfected normal mice Y = I.P. Challenge with 109 non-viable M. lepraemurium Z = I.P. Injection with 109 viable M. lepraemurium
2.4 16.3 16.9
6.2 2.2 5.9 1.9
46.5 13.3 14.8 19.1 46.5 22.0 5.6 25.5 39.6 11.2 22.9 20.6 51.8 5.0 11.5 29.4
503
Mycobacterial Infections
Vol. 68, No. 6
Table 3. SERUM PROTEIN ANALYSIS AND IMMUNOGLOBULIN ASSAY* -LEPROSY PATIENTS
Tuberculoid (11) Lepromatous (12) Borderline (5) Contacts (7) Healthy controls (4) *
=
Immunoglobulin
Serum Protein
Type
Prealb
Alb
A lpha
Beta
Gamma
IgA
IgG
-
59.98 53.8 59.94 70.07 60.17
20.05 15.2 15.10 10.24 17.65
14.0 10.73 17.94 11.44 17.55
5.78 18.62 6.5 8.07 4.62
290.0 546.0 314.0 ND
1577.1 2268.0 1628.0 ND 1000.0
-
220
IgM 156.0 279.6 206.4 ND 98.0
Values expressed in mg%
the results of the studies on humoral antibodies in patients.14 Immunoglobulin assays. These assays were carried out on sera from all groups of mice and those from patients. Table 4 gives the results of the immunoglobulin assay on M. leprae groups of mice. Compared to the normal values, the other groups show an increased level of all the classes of immunoglobulins viz., IgA, IgG1 and IgG2 and IgM. Among these IgA and IgG1 appear to increase as the infection progresses whereas the other two, IgG2 and IgM remain constant. Group A (infected) exhibits and increased level of IgG2 as well. Table 5 gives the data on serum immunoglobulins in mice infected or immunized with M. lepraemurium. The infected group (Group Z) shows elevated levels of all the classes of immunoglobulins compared to the other two groups. Among these IgA, and IgG1 seem to increase as the infection progresses while the other classes viz IgG2a, IgG.,b and IgM tend to remain constant. This is somewhat similar to the situation seen in mice infected with M. leprae. Immunoglobulin assays on sera from patients revealed that IgG increases as the clinical spectrum shifts from the early stages (TT) to advanced stages (LL), through borderline (BB), while the reverse is seen with IgA which decreases as the disease progresses. Increased IgM levels are noticed in sera from lepromatous patients as compared to those observed in sera from either the tuberculoid and borderline patients or from contacts of leprosy patients
and healthy individuals. These results are summarized in Table 3. DISCUSSION
Present studies were undertaken with a view to determining changes, in the serum proteins, and the levels of immunoglobulins of mice infected with M. leprae and M. lepraemurium and to compare them with those found in leprosy patients. Studies on serum proteins and immunoglobulins as well as the humoral and cell mediated immune response have been carried out by many investigators on patients in various stages of leprosy infection.5'8,'012'13'15 Since the time the mouse model has been recognized as a useful tool for the growth of M. leprae and for evaluation of anti-leprosy drugs, several investigations have been carried out on the immune response in mice infected with M. leprae.16-18 Previous work done in this laboratory on mice infected with M. leprae and M. lepraemurium on humoral as well as cell mediated immune response were found to be supportive of the view that the mouse model when infected with M. leprae could represent the early stage of human leprosy while the M. lepraemurium infection in mice could resemble an advanced stage of the disease. 10-13 Current studies were designed to see whether the serum protein changes and changes in the immunoglobulin levels also lend support to this contention. The agarase electrophoretic studies of serum protein analysis on sera from mice infected with
504
JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION
M. leprae, showed that the levels of gamma globulin in these mice are slightly higher than the normal values and that there is an increase in the gamma globulin level as the disease progresses. Similar situation is observed in mice infected with M. lepraemurium but the levels of gamma globulin are comparatively higher. Serum protein analysis of sera from patients with leprosy indicated that in early phase of the disease the gamma globulin level remains close to that seen in uninfected individuals. However, in the advanced stages of infection, the levels are rather high. Borderline cases and contacts fall within these two extremes. The results clearly indicate that the gamma globulin levels of mice infected with M. leprae are significantly similar to those observed in sera from tuberculoid leprosy patients while those of M. lepraemurium infected mice resemble the levels of gamma globulin observed in sera from lepromatous leprosy patients. The data obtained in the immunoglobulin assays have revealed increased immunoglobulin levels in mice treated with M. leprae and M. lepraemurium. Among the three classes of immunoglobulins studied the IgM levels were constant while the IgG and 1LA were seen to increase as the disease progressed. Between the two subclasses of IgG, the IgG, levels were seen to increase while IgG., remained constant. Immunoglobulin assay on sera from patients indicated that the levels of the three classes of immunoglobulins were higher than that of healthy controls. While IgG seems to increase proportionately with the progression of the disease from an early stage to the advanced stage; the IgA appears to decline, whereas, the IgM levels remain constant. The observations made in the current studies indicate a trend towards the placement of the mouse model infected with M. leprae in the early stage of leprosy infection in humans, whereas that infected with M. lepraemurium fits within the spectrum of the advanced stages of the disease. The data tend to support our previous ob-
"It0
NOVEMBER, 1976
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