[Frontiers in Bioscience 3, a58-65, November 1, 1998]

	[Frontiers in Bioscience 3, a58-65, November 1, 1998] Reprints PubMed CAVEAT LECTOR
	EARLY INDUCTION AND AUGMENTATION OF PARASITIC ANTIGEN-SPECIFIC ANTIBODY-PRODUCING B LYMPHOCYTES IN THE NON-PEYER’S PATCH REGION OF THE SMALL INTESTINE Ching-Hua Wang¹, Elizabeth M. Richards¹, Robert D. Block², Enio M. Lezcano¹ and Ricardo Gutierrez¹ 1 Biology Department, California State university, 5500 University parkway, San Bernardino, CA 92407, USA, ² Department of Neurology and Neural Science, School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received 10/12/98 Accepted 10/20/98 4. RESULTS To determine the kinetics of the appearance of 9D4 antigen-specific IgA-, IgG1-, IgG2a-, IgG2b-, IgG2c-, IgE-, and IgM-producing B lymphocytes (Ig-PC), SD rats were infected with 2,000 T. spiralis muscle larvae on day 0, and tissue samples were taken from the small intestine , Peyer’s patch, mesenteric lymph node, and the spleen on days 1, 2, 3, 5 and 7. Control samples were taken on days 0 and 5 from uninfected rats. Frozen sections of the tissues were double labeled with monoclonal mouse anti-rat specific immunoglobulin, followed by fluorescein (FITC)-conjugated goat anti-mouse nonspecific Ig- and rhodamine (XRITC)-conjugated monoclonal antibody affinity purified T. spiralis 9D4 antigen. The number of 9D4 antigen-specific Ig-PC was quantified by immunofluorescence microscopy. 4.1. Kinetics of appearance of 9D4 antigen-specific IgM-producing B lymphocytes The tissues described above were labeled with both XRITC-conjugated 9D4 antigen and the monoclonal mouse anti-rat IgM antibody plus the FITC-conjugated goat anti-mouse Ig to determine the number of 9D4 antigen-specific IgM-producing B cells. The results show (figure 1) that in the non-Peyer’s patch region of the small intestine, the number of 9D4-specific IgM-PC per VCU increased significantly from day 2 (22 + 3) onto day 5 (18 + 4) as compared to the pooled controls (3 + 2), with day 3 being the peak response (31 + 5). In the germinal centers of the Peyer’s patch, significant increase was detected from day 1 onto day 5, with days 1, 2, 3 and 5 showing 17 + 2, 21 + 2, 7 + 4, and 11 + 7 double labeled cells, respectively, as compared to the control of 0 + 1. Throughout the duration of examination, no significant increase in the number of 9D4-specific IgM-expressing B lymphocytes was observed in the non-germinal centers of the Peyer’s patch. The number was augmented significantly only on day 3 (11 + 7) for the MLN, and on days 5 (14 + 1) and 7 (4 + 1) for the spleen. Figure 1. Kinetics of appearance of 9D4-specific IgM-PC in rats infected with T. spiralis. Five groups of 3 rats were infected on day 0 with 2,000 muscle larvae orally and their intestinal (INT), mesenteric lymph node (MLN), Peyer’s patch (PP-GC: Peyer’s patch germinal center; PP-NGC: Peyer’s patch non-germinal center), and spleen (SPL) tissues were obtained on days 1, 2, 3, 5, or 7, respectively, with day 0 as the pooled uninfected controls. Same tissues of 3 of these uninfected rats were taken from day 0 and of another 3 taken on day 5. All the tissues were histologically processed and labeled first with monoclonal mouse-anti-rat IgM (H chain specific) antibody, and then with FITC-goat anti-mouse IgG (F(ab)’₂) and XRITC-9D4 antigen. Data represent mean numbers (with SD not shown) of 3 rats per infected group and 6 rats in the pooled control group. Comparing to the controls, significance (p<0.05) was only found in the number of IgM-PC in the intestine on days 2-5, in the MLN on day 3, in the PP-GC, on days 1-5, and in the spleen, on days 5 and 7. 4.2. Kinetics of appearance of 9D4 antigen-specific IgA-producing B lymphocytes When the same tissues were stained with both the XRITC-conjugated 9D4 antigen and the FITC-labeled goat anti-mouse antibody in addition to the monoclonal mouse anti-rat IgA antibody, insignificant results were obtained in tissues on almost all days examined with the exception of days 5 (32 + 9) and 7 (30 + 9) for the small intestine, and on the same days, both days 5 and 7, showing 5 + 1 9D4-specific IgM B cells in the spleen (figure 2). The Peyer’s patch revealed 9D4-specific IgA-PC in two different cell populations. Germinal center populations (PP-GC) contained some cells with immunoglobulins aggregated in clumps exhibiting a bright uneven signal, but showed insignificant numbers of IgA-PC to be present for the duration of the infection as mentioned above (0 + 0 on days 0 and 1, 9 + 5 on day 2, 7 + 2 on day 3, 5 + 2 on day 5, and 14 + 10 on day 7 of infection). In the non-germinal center regions (PP-NGC), one could barely detect any change in the infected rats from the pooled control throughout the experiment (0 + 0 IgA-PC on days, 0, 1, 2, 3 and 7, and 1 + 1 per field of vision on day 5 of infection). Similar results were found in the mesenteric lymph node (0 + 0 on days 0 and 3, 1 + 0 on day 2, and 0 + 1 per field of vision on days 1, 5 and 7 of infection). Figure 2. Kinetics of appearance of 9D4-specific IgA-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgA (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine and the spleen on days 5 and 7. 4.3. Kinetics of appearance of 9D4 antigen-specific IgE-producing B lymphocytes As shown in figure 3, the non Peyer’s patch region of the intestinal samples labeled with monoclonal mouse anti-rat IgE and 9D4 antigen showed a significant increase in 9D4-specific IgE-PC beginning on day 2 of infection (18 + 9 per VCU) and maintained the significant pattern of augmentation for the remainder of the experiment (16 + 9 on day 3, 45 + 9 on day 5, and 31 + 14 on day 7 of infection), as compared to the pooled control (1 + 3). Another significant increase was observed on day 5 of infection, as compared to day 3, whereas a significant decrease was observed on day 7 of infection, as compared to day 5 while maintaining significance from the pooled control. The Peyer’s patch germinal centers (figure 3) also showed significant numbers of 9D4-specific IgE-PC from day 2 onward (29 + 16 on day 2, 17 + 12 on day 3, and 12 + 5 and 18 + 8 for days 5 and 7, respectively), as compared to the pooled control (0 + 1). The non-germinal center regions of the Peyer’s patch failed to show any significant increase in IgE-PC throughout the duration of the experiment, as compared to the pooled control (0 + 1 on days 0 and 1, 1 + 1 on day 2, 2 + 0 on day 3, 2 + 1 on day 5, and 3 + 3 on day 7 of infection). Significant increase in 9D4-specific IgE-PC occurred in the mesenteric lymph node (figure 3) on days 2 (13 + 0) and 7 of infection (23 + 10 per field of vision), as compared to the pooled control (1 + 0), with day 5 (12 + 6) showing insignificant result. The spleen (figure 3) showed a significant increase in the mean number of antigen-specific IgE-PC from day 3 of infection (17 + 9 per field), and remained significant throughout the remainder of the experiment, as compared to the pooled control (0 + 1 on day 1, 20 + 16 on day 5, and 21 + 13 per field of vision on day 7 of infection). Figure 3. Kinetics of appearance of 9D4-specific IgE-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgE (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine and the PP-GC from day 2 through 7, in the MLN on days 2 and 7, and in the spleen from day 3 to 7. 4.4. Kinetics of appearance of 9D4 antigen-specific IgG1-producing B lymphocytes Intestinal samples taken from infected rats and labeled with monoclonal mouse anti-rat IgG1 and the 9D4 antigen showed a significant increase in 9D4-specific IgG1-PC as early as day 1 of infection (16 + 6). This increasing trend peaked on day 3 (44 + 10), as compared to the pooled control (0 + 0). Although it declined from the peak for the remaining period examined, but it was still significantly above the control (25 + 4 on day 5 and 31 + 12 per VCU on day 7 of infection, figure 4). The Peyer’s patch, though, only showed a significant increase of 9D4-specific IgG1-PC in the germinal center regions on day 5 of infection (18 + 7), as compared to the pooled control (0 + 1), and the number dropped to insignificance again on day 7 of infection (13 + 6 per field). Non-germinal center regions of the Peyer’s patch did not significantly differ from the pooled control (0 + 0) throughout the entire period examined (figure 4). In the mesenteric lymph node, a significant increase was first observed on day 3 (10 + 5), comparing to the pooled control (0 + 1). A significant decrease in 9D4-specific IgG1-PC numbers was then observed on day 5 of infection (3 + 4), as compared to day 3. However, day 7 showed another increase to a significant mean (11 + 4), as compared to the pooled control and day 5 of infection (figure 4). In the spleen (figure 4), significantly enhanced 9D4-specific IgG1-PC numbers were only detected on day 3 of infection (22 + 8) over that of the pooled control (2 + 4). Figure 4. Kinetics of appearance of 9D4-specific IgG1-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgG1 (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine from day 1 through 7, in the MLN on days 3 and 7, in the PP-GC on day 5 alone, and in the spleen on day 3 alone. 4.5. Kinetics of appearance of 9D4 antigen-specific IgG2a-producing B lymphocytes The results presented in figure 5 showed that the intestinal tissues labeled with monoclonal mouse-anti-rat IgG2a and the 9D4 antigen had a pooled control mean of 4 + 5 9D4-specific IgG2a-PC per VCU. On day 1, the number was 20 + 18, an insignificant result. Beginning on day 2, however, 24 + 8 of antigen-specific IgG2a-PC were enumerated per VCU, which was significantly higher than the control. Similar level of augmentation was revealed on the following days examined, with 23 + 7 on day 3, 21 + 9 on day 5, and 22 + 11 per VCU on day 7 of infection, all of which were significantly higher than the control. The Peyer’s patch germinal centers (figure 5) maintained significant numbers of 9D4-specific IgG2a-PC throughout the experiment, as compared to the control (0 + 0 on day 0, 7 + 2 on day 1, 17 + 7 on day 2, 16 + 2 on day 3, 13 + 4 on day 5, and 11 + 2 per field of vision on day 7 of infection). Non-germinal center of the Peyer’s patch showed insignificant numbers of 9D4-specific IgG2a-PC for the duration of the experiment, as compared to the pooled control (0 + 0 on days 0, 1, 3 and 7, 1 + 1 on day 2 and 1 + 2 per field of vision on day 5). Similar insignificant kinetics were found in the mesenteric lymph node (figure 5) throughout the duration of the experiment, with 5 + 4 on day 0, 7 + 2 on day 1, 6 + 1 on day 2, 5 + 4 on day 3, 6 + 3 on day 5, and 3 + 3 per field of vision on day 7 of infection. The spleen (figure 5) showed a significant mean number of antigen-specific IgG2a-PC on day 1 of infection (7 + 2 per field of vision), as compared to the pooled control (1 + 1). A significant decrease in mean number was observed by day 3 (2 + 1), as compared to that of day 1. This was followed by another significant increase on day 5 (19 + 1). Again, the mean number of 9D4-specific IgG2a-PC dropped on day 7 (6 + 2) but remained significant from the pooled control. Figure 5. Kinetics of appearance of 9D4-specific IgG2a-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgG2a (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine and the PP-GC from day 2 through 7, and in the spleen on days 1, 5 and 7. 4.6. Kinetics of appearance of 9D4 antigen-specific IgG2b-producing B lymphocytes The non-Peyer’s patch region of the small intestinal tissues labeled with monoclonal mouse anti-rat IgG2b and the 9D4 antigen contained a significantly increased number of the 9D4-specific IgG2c-PC per VCU on days 2 (42 + 25), 3 (37 + 6) and 7 (40 + 16) over the pooled control (4 + 3), with days 1 (18 + 15) and 5 (15 + 15) being insignificant due to the large variations (figure 6). The Peyer’s patch germinal centers (figure 6) showed significant numbers of 9D4-specific IgG2b-PC on days 5 (14 + 4) and 7 (13 + 10) of infection, as compared to the pooled control (0 + 0). The non-germinal center regions did not show any significant increase in the same cells for the duration of the experiment, as compared to the pooled control (0 + 0). The mesenteric lymph node (figure 6) harbored significantly increased numbers of 9D4-specific IgG2b-PC over the control (3 + 1) on days 3 (9 + 3) and 7 (10 + 1) only. There was a significant decline in the number of antigen-specific IgG2b-PC on day 5 (5 + 0). The spleen (figure 6) presented a significant increase in 9D4 directed IgG2b-PC on days 5 (16 + 14) and 7 (24 + 1) only, as compared to the pooled control (2 + 1). Figure 6. Kinetics of appearance of 9D4-specific IgG2b-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgG2b (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine from days 2, 3 and 7, in the MLN on days 3 and 7, in the PP-GC and the spleen on days 5 and 7. 4.7. Kinetics of appearance of 9D4 antigen-specific IgG2c-producing B lymphocytes When the non Peyer’s patch tissues of the small intestine were examined after they had been labeled with monoclonal mouse anti-rat IgG2c and the same antigen used throughout the experiment, a similar pattern of kinetics was observed. As early as 2 days after infection, the number of 9D4-specific IgG2c-PC jumped significantly (27 + 8) over the controls (2 + 3). Such significant proliferation continued to climb higher for the following days, with day 3 showing 33 + 3, day 5, 34 + 3 and day 7, 38 + 5 antigen-specific IgG2c-PC per VCU. No dip was observed throughout this period (figure 7). The germinal centers of the Peyer’s patch showed a significant increase on days 3 (15 + 11) through 5 after infection (14+7), followed by a decrease in mean number of 9D4-specific IgG2c-PC to insignificance by day 7 (5 + 1), as compared to the pooled control (1 + 1, figure 7). Once again, no significant change in kinetics of the numbers of 9D4-specific IgG2c-PC was demonstrated in the non-germinal center regions of the Peyer’s patch. The mesenteric lymph node showed a significant increase in these cells on days 2 (11 + 3) and 7 of infection (23 + 7), as compared to the pooled control (2 + 2). In between these two dates, a decline was observed (figure 7). No significant augmentation was demonstrated in the spleen until the last two days examined, with day 5 having 18 + 4 and day 7, 35 + 10 double labeled cells against the controls (0 + 0). Figure 7. Kinetics of appearance of 9D4-specific IgG2c-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgG2c (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine from day 2 through 7, in the MLN on days 2 and 7, in the PP-GC on days 3 and 5, and in the spleen on days 5 and 7. An example of the color signals produced by this labeling technique can be seen in figure 8, which contains photographs of samples of the non Peyer’s patch intestinal tissue labeled with monoclonal mouse anti-rat IgE primary antibody and the FITC-conjugated goat anti-rat Ig in combination with the XRITC-conjugated 9D4 antigen on day 5 of infection. Figure 8a shows the cells positively labeled with the FITC dye, indicating the IgE-PC, and figure 8b reveals the same cells in the identical field of examination showing the XRITC emission, indicating the 9D4 antigen-specificity of these cells. In both micrographs, the labeled cells were found in the lamina propria of the small intestine. Figure 8. Exhibition of 9D4 antigen-specific IgE-expressing cells in the lamina propria of the small intestine. The experimental procedures are described in figures 1 and 3. The intestinal tissue shown in these photographs was obtained from rat 5 days after infection with 2,000 muscle larvae of T. spiralis. The cryosection of the intestinal tissue was first labeled with monoclonal mouse anti-rat IgE (H chain specific) antibody and then, further labeled with FITC-goat anti mouse Ig (F(ab)'₂) and XRITC-9D4 antigen. Figure 8a, the one shows the green color, reveals IgE-producing cells labeled with the FITC dye in one villus crypt unit (VCU). Almost all the labeled cells occur in the lamina propria of the intestinal tissue. Figure 8b, the one shows the red color, demonstrates that the same cells in the identical VCU labeled with the FITC dye were also labeled with the XRITC dye, indicating that these cells were expressing IgE antibody molecules on the surface membrane that were specifically binding the 9D4 antigen. The size of the labeled cells range from 12 to 16 um in diameter.

[Frontiers in Bioscience 3, a58-65, November 1, 1998]
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PubMed
CAVEAT LECTOR

EARLY INDUCTION AND AUGMENTATION OF PARASITIC ANTIGEN-SPECIFIC ANTIBODY-PRODUCING B LYMPHOCYTES IN THE NON-PEYER’S PATCH REGION OF THE SMALL INTESTINE

Ching-Hua Wang¹, Elizabeth M. Richards¹, Robert D. Block², Enio M. Lezcano¹ and Ricardo Gutierrez¹

1 Biology Department, California State university, 5500 University parkway, San Bernardino, CA 92407, USA, ² Department of Neurology and Neural Science, School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Received 10/12/98 Accepted 10/20/98

4. RESULTS

To determine the kinetics of the appearance of 9D4 antigen-specific IgA-, IgG1-, IgG2a-, IgG2b-, IgG2c-, IgE-, and IgM-producing B lymphocytes (Ig-PC), SD rats were infected with 2,000 T. spiralis muscle larvae on day 0, and tissue samples were taken from the small intestine , Peyer’s patch, mesenteric lymph node, and the spleen on days 1, 2, 3, 5 and 7. Control samples were taken on days 0 and 5 from uninfected rats. Frozen sections of the tissues were double labeled with monoclonal mouse anti-rat specific immunoglobulin, followed by fluorescein (FITC)-conjugated goat anti-mouse nonspecific Ig- and rhodamine (XRITC)-conjugated monoclonal antibody affinity purified T. spiralis 9D4 antigen. The number of 9D4 antigen-specific Ig-PC was quantified by immunofluorescence microscopy.

4.1. Kinetics of appearance of 9D4 antigen-specific IgM-producing B lymphocytes

The tissues described above were labeled with both XRITC-conjugated 9D4 antigen and the monoclonal mouse anti-rat IgM antibody plus the FITC-conjugated goat anti-mouse Ig to determine the number of 9D4 antigen-specific IgM-producing B cells. The results show (figure 1) that in the non-Peyer’s patch region of the small intestine, the number of 9D4-specific IgM-PC per VCU increased significantly from day 2 (22 + 3) onto day 5 (18 + 4) as compared to the pooled controls (3 + 2), with day 3 being the peak response (31 + 5). In the germinal centers of the Peyer’s patch, significant increase was detected from day 1 onto day 5, with days 1, 2, 3 and 5 showing 17 + 2, 21 + 2, 7 + 4, and 11 + 7 double labeled cells, respectively, as compared to the control of 0 + 1. Throughout the duration of examination, no significant increase in the number of 9D4-specific IgM-expressing B lymphocytes was observed in the non-germinal centers of the Peyer’s patch. The number was augmented significantly only on day 3 (11 + 7) for the MLN, and on days 5 (14 + 1) and 7 (4 + 1) for the spleen.

Figure 1. Kinetics of appearance of 9D4-specific IgM-PC in rats infected with T. spiralis. Five groups of 3 rats were infected on day 0 with 2,000 muscle larvae orally and their intestinal (INT), mesenteric lymph node (MLN), Peyer’s patch (PP-GC: Peyer’s patch germinal center; PP-NGC: Peyer’s patch non-germinal center), and spleen (SPL) tissues were obtained on days 1, 2, 3, 5, or 7, respectively, with day 0 as the pooled uninfected controls. Same tissues of 3 of these uninfected rats were taken from day 0 and of another 3 taken on day 5. All the tissues were histologically processed and labeled first with monoclonal mouse-anti-rat IgM (H chain specific) antibody, and then with FITC-goat anti-mouse IgG (F(ab)’₂) and XRITC-9D4 antigen. Data represent mean numbers (with SD not shown) of 3 rats per infected group and 6 rats in the pooled control group. Comparing to the controls, significance (p<0.05) was only found in the number of IgM-PC in the intestine on days 2-5, in the MLN on day 3, in the PP-GC, on days 1-5, and in the spleen, on days 5 and 7.

4.2. Kinetics of appearance of 9D4 antigen-specific IgA-producing B lymphocytes

When the same tissues were stained with both the XRITC-conjugated 9D4 antigen and the FITC-labeled goat anti-mouse antibody in addition to the monoclonal mouse anti-rat IgA antibody, insignificant results were obtained in tissues on almost all days examined with the exception of days 5 (32 + 9) and 7 (30 + 9) for the small intestine, and on the same days, both days 5 and 7, showing 5 + 1 9D4-specific IgM B cells in the spleen (figure 2). The Peyer’s patch revealed 9D4-specific IgA-PC in two different cell populations. Germinal center populations (PP-GC) contained some cells with immunoglobulins aggregated in clumps exhibiting a bright uneven signal, but showed insignificant numbers of IgA-PC to be present for the duration of the infection as mentioned above (0 + 0 on days 0 and 1, 9 + 5 on day 2, 7 + 2 on day 3, 5 + 2 on day 5, and 14 + 10 on day 7 of infection). In the non-germinal center regions (PP-NGC), one could barely detect any change in the infected rats from the pooled control throughout the experiment (0 + 0 IgA-PC on days, 0, 1, 2, 3 and 7, and 1 + 1 per field of vision on day 5 of infection). Similar results were found in the mesenteric lymph node (0 + 0 on days 0 and 3, 1 + 0 on day 2, and 0 + 1 per field of vision on days 1, 5 and 7 of infection).

Figure 2. Kinetics of appearance of 9D4-specific IgA-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgA (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine and the spleen on days 5 and 7.

4.3. Kinetics of appearance of 9D4 antigen-specific IgE-producing B lymphocytes

As shown in figure 3, the non Peyer’s patch region of the intestinal samples labeled with monoclonal mouse anti-rat IgE and 9D4 antigen showed a significant increase in 9D4-specific IgE-PC beginning on day 2 of infection (18 + 9 per VCU) and maintained the significant pattern of augmentation for the remainder of the experiment (16 + 9 on day 3, 45 + 9 on day 5, and 31 + 14 on day 7 of infection), as compared to the pooled control (1 + 3). Another significant increase was observed on day 5 of infection, as compared to day 3, whereas a significant decrease was observed on day 7 of infection, as compared to day 5 while maintaining significance from the pooled control. The Peyer’s patch germinal centers (figure 3) also showed significant numbers of 9D4-specific IgE-PC from day 2 onward (29 + 16 on day 2, 17 + 12 on day 3, and 12 + 5 and 18 + 8 for days 5 and 7, respectively), as compared to the pooled control (0 + 1). The non-germinal center regions of the Peyer’s patch failed to show any significant increase in IgE-PC throughout the duration of the experiment, as compared to the pooled control (0 + 1 on days 0 and 1, 1 + 1 on day 2, 2 + 0 on day 3, 2 + 1 on day 5, and 3 + 3 on day 7 of infection). Significant increase in 9D4-specific IgE-PC occurred in the mesenteric lymph node (figure 3) on days 2 (13 + 0) and 7 of infection (23 + 10 per field of vision), as compared to the pooled control (1 + 0), with day 5 (12 + 6) showing insignificant result. The spleen (figure 3) showed a significant increase in the mean number of antigen-specific IgE-PC from day 3 of infection (17 + 9 per field), and remained significant throughout the remainder of the experiment, as compared to the pooled control (0 + 1 on day 1, 20 + 16 on day 5, and 21 + 13 per field of vision on day 7 of infection).

Figure 3. Kinetics of appearance of 9D4-specific IgE-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgE (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine and the PP-GC from day 2 through 7, in the MLN on days 2 and 7, and in the spleen from day 3 to 7.

4.4. Kinetics of appearance of 9D4 antigen-specific IgG1-producing B lymphocytes

Intestinal samples taken from infected rats and labeled with monoclonal mouse anti-rat IgG1 and the 9D4 antigen showed a significant increase in 9D4-specific IgG1-PC as early as day 1 of infection (16 + 6). This increasing trend peaked on day 3 (44 + 10), as compared to the pooled control (0 + 0). Although it declined from the peak for the remaining period examined, but it was still significantly above the control (25 + 4 on day 5 and 31 + 12 per VCU on day 7 of infection, figure 4). The Peyer’s patch, though, only showed a significant increase of 9D4-specific IgG1-PC in the germinal center regions on day 5 of infection (18 + 7), as compared to the pooled control (0 + 1), and the number dropped to insignificance again on day 7 of infection (13 + 6 per field). Non-germinal center regions of the Peyer’s patch did not significantly differ from the pooled control (0 + 0) throughout the entire period examined (figure 4). In the mesenteric lymph node, a significant increase was first observed on day 3 (10 + 5), comparing to the pooled control (0 + 1). A significant decrease in 9D4-specific IgG1-PC numbers was then observed on day 5 of infection (3 + 4), as compared to day 3. However, day 7 showed another increase to a significant mean (11 + 4), as compared to the pooled control and day 5 of infection (figure 4). In the spleen (figure 4), significantly enhanced 9D4-specific IgG1-PC numbers were only detected on day 3 of infection (22 + 8) over that of the pooled control (2 + 4).

Figure 4. Kinetics of appearance of 9D4-specific IgG1-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgG1 (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine from day 1 through 7, in the MLN on days 3 and 7, in the PP-GC on day 5 alone, and in the spleen on day 3 alone.

4.5. Kinetics of appearance of 9D4 antigen-specific IgG2a-producing B lymphocytes

The results presented in figure 5 showed that the intestinal tissues labeled with monoclonal mouse-anti-rat IgG2a and the 9D4 antigen had a pooled control mean of 4 + 5 9D4-specific IgG2a-PC per VCU. On day 1, the number was 20 + 18, an insignificant result. Beginning on day 2, however, 24 + 8 of antigen-specific IgG2a-PC were enumerated per VCU, which was significantly higher than the control. Similar level of augmentation was revealed on the following days examined, with 23 + 7 on day 3, 21 + 9 on day 5, and 22 + 11 per VCU on day 7 of infection, all of which were significantly higher than the control. The Peyer’s patch germinal centers (figure 5) maintained significant numbers of 9D4-specific IgG2a-PC throughout the experiment, as compared to the control (0 + 0 on day 0, 7 + 2 on day 1, 17 + 7 on day 2, 16 + 2 on day 3, 13 + 4 on day 5, and 11 + 2 per field of vision on day 7 of infection). Non-germinal center of the Peyer’s patch showed insignificant numbers of 9D4-specific IgG2a-PC for the duration of the experiment, as compared to the pooled control (0 + 0 on days 0, 1, 3 and 7, 1 + 1 on day 2 and 1 + 2 per field of vision on day 5). Similar insignificant kinetics were found in the mesenteric lymph node (figure 5) throughout the duration of the experiment, with 5 + 4 on day 0, 7 + 2 on day 1, 6 + 1 on day 2, 5 + 4 on day 3, 6 + 3 on day 5, and 3 + 3 per field of vision on day 7 of infection. The spleen (figure 5) showed a significant mean number of antigen-specific IgG2a-PC on day 1 of infection (7 + 2 per field of vision), as compared to the pooled control (1 + 1). A significant decrease in mean number was observed by day 3 (2 + 1), as compared to that of day 1. This was followed by another significant increase on day 5 (19 + 1). Again, the mean number of 9D4-specific IgG2a-PC dropped on day 7 (6 + 2) but remained significant from the pooled control.

Figure 5. Kinetics of appearance of 9D4-specific IgG2a-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgG2a (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine and the PP-GC from day 2 through 7, and in the spleen on days 1, 5 and 7.

4.6. Kinetics of appearance of 9D4 antigen-specific IgG2b-producing B lymphocytes

The non-Peyer’s patch region of the small intestinal tissues labeled with monoclonal mouse anti-rat IgG2b and the 9D4 antigen contained a significantly increased number of the 9D4-specific IgG2c-PC per VCU on days 2 (42 + 25), 3 (37 + 6) and 7 (40 + 16) over the pooled control (4 + 3), with days 1 (18 + 15) and 5 (15 + 15) being insignificant due to the large variations (figure 6). The Peyer’s patch germinal centers (figure 6) showed significant numbers of 9D4-specific IgG2b-PC on days 5 (14 + 4) and 7 (13 + 10) of infection, as compared to the pooled control (0 + 0). The non-germinal center regions did not show any significant increase in the same cells for the duration of the experiment, as compared to the pooled control (0 + 0). The mesenteric lymph node (figure 6) harbored significantly increased numbers of 9D4-specific IgG2b-PC over the control (3 + 1) on days 3 (9 + 3) and 7 (10 + 1) only. There was a significant decline in the number of antigen-specific IgG2b-PC on day 5 (5 + 0). The spleen (figure 6) presented a significant increase in 9D4 directed IgG2b-PC on days 5 (16 + 14) and 7 (24 + 1) only, as compared to the pooled control (2 + 1).

Figure 6. Kinetics of appearance of 9D4-specific IgG2b-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgG2b (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine from days 2, 3 and 7, in the MLN on days 3 and 7, in the PP-GC and the spleen on days 5 and 7.

4.7. Kinetics of appearance of 9D4 antigen-specific IgG2c-producing B lymphocytes

When the non Peyer’s patch tissues of the small intestine were examined after they had been labeled with monoclonal mouse anti-rat IgG2c and the same antigen used throughout the experiment, a similar pattern of kinetics was observed. As early as 2 days after infection, the number of 9D4-specific IgG2c-PC jumped significantly (27 + 8) over the controls (2 + 3). Such significant proliferation continued to climb higher for the following days, with day 3 showing 33 + 3, day 5, 34 + 3 and day 7, 38 + 5 antigen-specific IgG2c-PC per VCU. No dip was observed throughout this period (figure 7). The germinal centers of the Peyer’s patch showed a significant increase on days 3 (15 + 11) through 5 after infection (14+7), followed by a decrease in mean number of 9D4-specific IgG2c-PC to insignificance by day 7 (5 + 1), as compared to the pooled control (1 + 1, figure 7). Once again, no significant change in kinetics of the numbers of 9D4-specific IgG2c-PC was demonstrated in the non-germinal center regions of the Peyer’s patch. The mesenteric lymph node showed a significant increase in these cells on days 2 (11 + 3) and 7 of infection (23 + 7), as compared to the pooled control (2 + 2). In between these two dates, a decline was observed (figure 7). No significant augmentation was demonstrated in the spleen until the last two days examined, with day 5 having 18 + 4 and day 7, 35 + 10 double labeled cells against the controls (0 + 0).

Figure 7. Kinetics of appearance of 9D4-specific IgG2c-PC in rats infected with T. spiralis. The experimental procedure is as described in figure 1, except the primary antibody used was monoclonal mouse anti-rat IgG2c (H chain specific). Comparing to the controls, significance (p<0.05) was only found in the intestine from day 2 through 7, in the MLN on days 2 and 7, in the PP-GC on days 3 and 5, and in the spleen on days 5 and 7.

An example of the color signals produced by this labeling technique can be seen in figure 8, which contains photographs of samples of the non Peyer’s patch intestinal tissue labeled with monoclonal mouse anti-rat IgE primary antibody and the FITC-conjugated goat anti-rat Ig in combination with the XRITC-conjugated 9D4 antigen on day 5 of infection. Figure 8a shows the cells positively labeled with the FITC dye, indicating the IgE-PC, and figure 8b reveals the same cells in the identical field of examination showing the XRITC emission, indicating the 9D4 antigen-specificity of these cells. In both micrographs, the labeled cells were found in the lamina propria of the small intestine.

Figure 8. Exhibition of 9D4 antigen-specific IgE-expressing cells in the lamina propria of the small intestine. The experimental procedures are described in figures 1 and 3. The intestinal tissue shown in these photographs was obtained from rat 5 days after infection with 2,000 muscle larvae of T. spiralis. The cryosection of the intestinal tissue was first labeled with monoclonal mouse anti-rat IgE (H chain specific) antibody and then, further labeled with FITC-goat anti mouse Ig (F(ab)'₂) and XRITC-9D4 antigen. Figure 8a, the one shows the green color, reveals IgE-producing cells labeled with the FITC dye in one villus crypt unit (VCU). Almost all the labeled cells occur in the lamina propria of the intestinal tissue. Figure 8b, the one shows the red color, demonstrates that the same cells in the identical VCU labeled with the FITC dye were also labeled with the XRITC dye, indicating that these cells were expressing IgE antibody molecules on the surface membrane that were specifically binding the 9D4 antigen. The size of the labeled cells range from 12 to 16 um in diameter.