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ACV with/without IVM: a new talk on intestinal CDX2 and muscular CD34 and Cyclin D1 during Trichinella spiralis infection

E. A. El Saftawy
E. A. El Saftawy
, 
B. E. Aboulhoda
B. E. Aboulhoda
, 
F. E. Hassan
F. E. Hassan
, 
M. A. M. Ismail
M. A. M. Ismail
, 
M. A. Alghamdi
M. A. Alghamdi
, 
S. M. Hussein
S. M. Hussein
 oraz 
N. M. Amin
N. M. Amin
   | 16 lip 2024
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Data publikacji: 16 lip 2024
Zakres stron: 124 - 141
Otrzymano: 04 sty 2024
Przyjęty: 25 mar 2024
DOI: https://doi.org/10.2478/helm-2024-0013
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© 2024 E. A. El Saftawy et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Experimental design.
Experimental design.

Fig. 2.

Photomicrographs of H&E-stained small intestine in infected and untreated mice at day 7p.i.. A, B, & C show adults’ cut sections invading the core and base of the intestinal villi in three different tissue cut sections surrounded with dense inflammatory infiltrates. Yet, (A) shows a reduced V length/C depth ratio, flattening villi, and shedding of epithelial cells (C) shows increased Paneth cells with eosinophilic cytoplasm, (D) shows hyperplasia of goblet cells, (E) shows increased inflammatory cells in the neighboring villi, (F) shows flattening and hyperplasia of the crypts of the villi (red arrow), disrupted villi (yellow arrow), and dense inflammatory cells (black arrows).
Photomicrographs of H&E-stained small intestine in infected and untreated mice at day 7p.i.. A, B, & C show adults’ cut sections invading the core and base of the intestinal villi in three different tissue cut sections surrounded with dense inflammatory infiltrates. Yet, (A) shows a reduced V length/C depth ratio, flattening villi, and shedding of epithelial cells (C) shows increased Paneth cells with eosinophilic cytoplasm, (D) shows hyperplasia of goblet cells, (E) shows increased inflammatory cells in the neighboring villi, (F) shows flattening and hyperplasia of the crypts of the villi (red arrow), disrupted villi (yellow arrow), and dense inflammatory cells (black arrows).

Fig. 3.

Photomicrographs of H&E-stained small intestine in infected and treated mice at day 7 p.i.. (A.1) IVM treated mice, showing finger-like villi with apparently intact epithelial lining (yellow arrow), moderate inflammatory infiltrates (black arrow), and few disruptions in the surface of the villi. (A.2 and A.3) show goblet and Paneth cells respectively (black arrows). (B.1) ACV-treated mice, showing relatively intact epithelium lining with scarce disruption and reduced inflammatory infiltrates in finger-like villi (black arrows). (B.2 and B.3) show goblet and Paneth cells respectively (black arrows). (C.1) Combined treatment (IVM+ACV) showing finger-like and tongue-like villi with a mild intensity of the inflammatory infiltrate (black arrow), intact epithelium lining (yellow arrow). (C.2 and C.3) show few discrete goblets and Paneth cells respectively (black arrows). (D) Healthy control.
Photomicrographs of H&E-stained small intestine in infected and treated mice at day 7 p.i.. (A.1) IVM treated mice, showing finger-like villi with apparently intact epithelial lining (yellow arrow), moderate inflammatory infiltrates (black arrow), and few disruptions in the surface of the villi. (A.2 and A.3) show goblet and Paneth cells respectively (black arrows). (B.1) ACV-treated mice, showing relatively intact epithelium lining with scarce disruption and reduced inflammatory infiltrates in finger-like villi (black arrows). (B.2 and B.3) show goblet and Paneth cells respectively (black arrows). (C.1) Combined treatment (IVM+ACV) showing finger-like and tongue-like villi with a mild intensity of the inflammatory infiltrate (black arrow), intact epithelium lining (yellow arrow). (C.2 and C.3) show few discrete goblets and Paneth cells respectively (black arrows). (D) Healthy control.

Fig. 4.

Photomicrographs of H&E-stained muscle cut sections in infected untreated mice at day 35 p.i.. (A) Spiral morphology of larva (red arrow), nurse cell (blue arrow), capsule (black arrow), and atrophied muscle fibers. (B) The detailed structure of the encysted larva: stichocyte (S), hypertrophied nucleus (N). Note the septum that separates the parasite and nurse cell. (C) Mature nurse cells with eosinophilic non-homogeneous cytoplasm (blue arrow), collagen capsule (C) and intense inflammatory infiltrates (Inf), and satellite cells (under the basal lamina of the nurse cell) (D) hypertrophied nuclei (N) invading a nurse cell. (E) immature reproductive tract (R), lateral cord (L), intestinal tract (I), and blue arrow refers to nurse cell basal lamina (F) A cluster of larvae with intense inflammatory cells.
Photomicrographs of H&E-stained muscle cut sections in infected untreated mice at day 35 p.i.. (A) Spiral morphology of larva (red arrow), nurse cell (blue arrow), capsule (black arrow), and atrophied muscle fibers. (B) The detailed structure of the encysted larva: stichocyte (S), hypertrophied nucleus (N). Note the septum that separates the parasite and nurse cell. (C) Mature nurse cells with eosinophilic non-homogeneous cytoplasm (blue arrow), collagen capsule (C) and intense inflammatory infiltrates (Inf), and satellite cells (under the basal lamina of the nurse cell) (D) hypertrophied nuclei (N) invading a nurse cell. (E) immature reproductive tract (R), lateral cord (L), intestinal tract (I), and blue arrow refers to nurse cell basal lamina (F) A cluster of larvae with intense inflammatory cells.

Fig. 5.

Photomicrographs of H&E-stained muscle sections in infected and treated mice at day 35 p.i.. A, B, and C show the burden of larvae in treated groups. In D, the IVM-treated group showed nurse cells with scanty and slightly basophilic cytoplasm (blue arrow), moderate inflammatory infiltrates (black arrow), a remnant of larvae (red arrow), and atrophied unoccupied muscle fibers. In E, the ACV-treated group showed nurse cells with eosinophilic cytoplasm (blue arrow), mild to moderate inflammatory infiltrates (black arrow), variably damaged larvae (red arrows), and atrophied unoccupied muscle fibers. In (F) Combined treatment shows inflammatory infiltrates that partially invade the loose and vacuolated capsule (black arrow), homogenized larva lacking details (red arrow), regenerated muscle fibers (yellow arrows), and absence of muscle atrophy, while nurse cell was almost lacking.
Photomicrographs of H&E-stained muscle sections in infected and treated mice at day 35 p.i.. A, B, and C show the burden of larvae in treated groups. In D, the IVM-treated group showed nurse cells with scanty and slightly basophilic cytoplasm (blue arrow), moderate inflammatory infiltrates (black arrow), a remnant of larvae (red arrow), and atrophied unoccupied muscle fibers. In E, the ACV-treated group showed nurse cells with eosinophilic cytoplasm (blue arrow), mild to moderate inflammatory infiltrates (black arrow), variably damaged larvae (red arrows), and atrophied unoccupied muscle fibers. In (F) Combined treatment shows inflammatory infiltrates that partially invade the loose and vacuolated capsule (black arrow), homogenized larva lacking details (red arrow), regenerated muscle fibers (yellow arrows), and absence of muscle atrophy, while nurse cell was almost lacking.

Fig. 6.

Photomicrographs of immunohistochemical expression of CDX2 in the epithelial cells of the villi and crypts at day 7 p.i.. (A–D) Infected untreated mice show overexpression of nuclear and cytoplasmic CDX2. Note in (B–D) adult T. spiralis embedded in the villi (red arrows). E and F show reduced expression of CDX2 in IVM- and ACV-treated mice respectively. G, combined treatment shows mainly nuclear staining for CDX2. H, healthy control.
Photomicrographs of immunohistochemical expression of CDX2 in the epithelial cells of the villi and crypts at day 7 p.i.. (A–D) Infected untreated mice show overexpression of nuclear and cytoplasmic CDX2. Note in (B–D) adult T. spiralis embedded in the villi (red arrows). E and F show reduced expression of CDX2 in IVM- and ACV-treated mice respectively. G, combined treatment shows mainly nuclear staining for CDX2. H, healthy control.

Fig. 7.

Photomicrograph showing immunohistochemical expression of Cyclin D1 in the muscle phase (day 35 p.i.). A, Infected untreated animals show intense cytoplasmic and nuclear expression of Cyclin D1 in the nurse cell (N), satellite cells (S), and the surrounding muscle fibers (M). B and C show expression of CyclinD1 in unoccupied muscle fibers and clusters of encysted larvae respectively in infected untreated mice. Expression of Cyclin D1 declined in IVM (D–F) and ACV (G–I) treated groups in the capsules (red arrow), nurse cells (blue arrow), and unoccupied muscle fibers. J–L, combined treatment shows a profound reduction in CyclinD1 in both nurse cell remnants and unoccupied muscle fibers.
Photomicrograph showing immunohistochemical expression of Cyclin D1 in the muscle phase (day 35 p.i.). A, Infected untreated animals show intense cytoplasmic and nuclear expression of Cyclin D1 in the nurse cell (N), satellite cells (S), and the surrounding muscle fibers (M). B and C show expression of CyclinD1 in unoccupied muscle fibers and clusters of encysted larvae respectively in infected untreated mice. Expression of Cyclin D1 declined in IVM (D–F) and ACV (G–I) treated groups in the capsules (red arrow), nurse cells (blue arrow), and unoccupied muscle fibers. J–L, combined treatment shows a profound reduction in CyclinD1 in both nurse cell remnants and unoccupied muscle fibers.

Fig. 8.

Photomicrograph showing immunohistochemical expression of CD34 in the muscle phase (day 35 p.i.). (A) The infected untreated group shows missing or reduced CD34 localized between the individual muscle fibers. The black arrow refers to localized CD34 in mononuclear cells. The IVM-treated mice (B) and the ACV-treated group (C) show a moderate increase in the expression of CD34. (D) combined treatment shows intensification in the transmembrane expression of CD34 in association with remnants of nurse cells, damaged larva, and scarce cellular infiltrates. Note yellow arrow refers to immune reactive satellite cells. The black arrow refers to the capsule; the red arrow refers to the larva; and the blue block points to the remnants of the nurse cell.
Photomicrograph showing immunohistochemical expression of CD34 in the muscle phase (day 35 p.i.). (A) The infected untreated group shows missing or reduced CD34 localized between the individual muscle fibers. The black arrow refers to localized CD34 in mononuclear cells. The IVM-treated mice (B) and the ACV-treated group (C) show a moderate increase in the expression of CD34. (D) combined treatment shows intensification in the transmembrane expression of CD34 in association with remnants of nurse cells, damaged larva, and scarce cellular infiltrates. Note yellow arrow refers to immune reactive satellite cells. The black arrow refers to the capsule; the red arrow refers to the larva; and the blue block points to the remnants of the nurse cell.

Fig. 9.

Graphs of Spearman correlation. (A) Positive correlation between intestinal CDX2 O.D. (day 7 p.i.) and muscular Cyclin D1 expression (day 35 p.i.), (B) negative correlation between intestinal CDX2 (day 7 p.i.) and muscular CD34 (day 35 p.i.), and (C) negative correlation between muscular CD34 and Cyclin D1(day 35 p.i.).
Graphs of Spearman correlation. (A) Positive correlation between intestinal CDX2 O.D. (day 7 p.i.) and muscular Cyclin D1 expression (day 35 p.i.), (B) negative correlation between intestinal CDX2 (day 7 p.i.) and muscular CD34 (day 35 p.i.), and (C) negative correlation between muscular CD34 and Cyclin D1(day 35 p.i.).

Means of body weight and survival rate in all experimental subgroups.

Group 1 (infected - untreated) Group 2 (IVM treated) Group 3 (ACV treated) Group 4 (combined treatment) Group 5 (healthy) P-value
Mo SD Mo SD Mo SD Mo SD Mo SD
Body weight (g) at day 7 p.i. (subgroups, A) 15.70a 1.64 22.90b 2.13 23.10b 2.23 26.20c 1.03 26.60c 1.43 < 0.001
Body weight (g) at day 35 p.i. (subgroups, B) 18.20a 2.20 22.30b 2.11 23.70b 2.41 27.00c 1.25 27.30c 1.64 < 0.001
Survival rate 11a 73.3% 12a,b 80.0% 12a,b 80.0% 14a,b 93.3% 15b 100.0%

Mean and standard deviation of larva burden/LPF on day 35 p.i. (chronic stage).

Subgroup 1B (infected untreated) Subgroup 2B (IVM treated) Subgroup 3B (ACV treated) Subgroup 4B (combined treatment) Subgroup 5B (healthy) P-value
Mo SD Mo SD Mo SD Mo SD Mo SD
Larva burden/LPF 21.20a 5.73 6.80b 2.53 6.50b 2.22 2.70b 1.16 NA . < 0.001

Counts of Trichinella spiralis adult worms in enteric fluid and intestinal tissue on day 7 p.i.

Subgroup 1A (infected untreated) Subgroup 2A (IVM treated) Subgroup 3 A (ACV treated) Subgroup 4A (combined treatment) Subgroup 5A (healthy) P-value
Mo SD Mo SD R% Mo SD R% Mo SD R% Mo SD
No. of adults / milliliter 173.2a 65 46.2b 8.57 73.3% 45.6b 10.25 73.7% 21.7b 8.82 87.5% NA . < 0.001
No. of adults / 100 villi 30.1a 14 12b 5.08 60.1% 10.10b,c 3.93 66.5% 1.8c 1.32 94.02% NA . < 0.001

Muscular histopathological changes on day 35 p.i. (chronic stage).

Subgroup 1B (infected untreated) Subgroup 2B (IVM treated) Subgroup 3B (ACV treated) Subgroup 4B (combined treatment) Subgroup 5B (healthy) P-value
Count % Count % Count % Count % Count %
Larva structures Intact 10a 100.0% 5b 50.0% 6b 60.0% 0c 0.0% 0 0.0% < 0.001
Altered 0a 0.0% 5b 50.0% 4b 40.0% 10c 100.0% 0 0.0%
Capsule inflammatory infiltration Mild 0a 0.0% 2a,b 20.0% 5b,c 50.0% 9c 90.0% 0 0.0% < 0.001
moderate 3a,b 30.0% 6b 60.0% 5a,b 50.0% 1a 10.0% 0 0.0%
intense 7a 70.0% 2b 20.0% 0b 0.0% 0b 0.0% 0 0.0%
Nurse cell intact 10a 100.0% 4b 40.0% 5b 50.0% 2b 20.0% 0 0.0% 0.001
altered 0a 0.0% 6b 60.0% 5b 50.0% 8b 80.0% 0 0.0%

Intestinal histopathological changes on day 7 p.i. (acute stage).

Subgroup 1A (infected untreated) Subgroup 2A (IVM treated) Subgroup 3 A (ACV treated) Subgroup4 A (combined treatment) Subgroup 5A (healthy) P-value
Mo SD Mo SD Mo SD Mo SD Mo SD
V length /C depth ratio 1.39a 0.24 5.07b 0.80 4.87b 0.93 5.07b 0.80 5.60b 1.07 < 0.001
lymphocyte counts/villus 110.00a 13.03 50.20b 8.35 18.80c 4.26 6.60d 1.78 6.50d 1.84 < 0.001
Goblet cell counts /villus 45.50a 5.02 12.40b 2.27 12.40b 2.32 10.70 b,c 1.57 8.60c 0.97 < 0.001
No. of Paneth cells/crypt 16.90a 2.18 6.60b 0.97 6.00b 0.82 5.70b 0.82 5.70b 1.49 < 0.001
No. of crypts /villus 8.30a 1.89 2.30b 0.82 2.40b 0.70 1.60b 0.70 1.50b 0.53 < 0.001

Correlation of CDX2, Cyclin D1, and CD34 expressions with body weight, intestinal inflammation, and parasite burden.

CDX2 Cyclin D1 CD34
Body weight (g) at day 7 p.i. Correlation Coefficient, r-value -0.717- -0.853- 0.727
p-value < 0.001 < 0.001 < 0.001
No. 50 50 50
Body weight (g) at day 35 p.i. Correlation Coefficient, r-value -0.684- -0.829- 0.746
p-value < 0.001 < 0.001 < 0.001
No. 50 50 50
V length /C depth ratio Correlation Coefficient, r-value -0.496- -0.609- 0.647
p-value < 0.001 < 0.001 < 0.001
No. 50 50 50
lymphocyte counts/villus Correlation Coefficient, r-value 0.759 0.896 -0.799-
p-value < 0.001 < 0.001 < 0.001
No. 50 50 50
Goblet cell counts /villus Correlation Coefficient, r-value 0.731 0.761 -0.718-
p-value < 0.001 < 0.001 < 0.001
No. 50 50 50
No. of Paneth cells/crypt Correlation Coefficient, r-value 0.550 0.645 -0.619-
p-value < 0.001 < 0.001 < 0.001
No. 50 50 50
No. of crypts /villus Correlation Coefficient, r-value 0.644 0.714 -0.685-
p-value < 0.001 < 0.001 < 0.001
No. 50 50 50
Adult burden/milliliter at day 7 p.i. Correlation Coefficient, r-value 0.783 0.834 -0.755-
p-value < 0.001 < 0.001 < 0.001
No. 40 40 40
Adult burden/100 villi at day 7 p.i. Correlation Coefficient, r-value 0.770 0.837 -0.736-
p-value < 0.001 < 0.001 < 0.001
No. 40 40 40
larva burden/LPF at day 35 p.i. Correlation Coefficient, r-value 0.690 0.799 -0.802-
p-value < 0.001 < 0.001 < 0.001
No. 40 40 40
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Język:
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Częstotliwość wydawania:
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Dziedziny czasopisma:
Life Sciences, Zoology, Ecology, other, Medicine, Clinical Medicine, Microbiology, Virology and Infection Epidemiology
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