Following ingestion of the infective larvae, it establishes a distinctive intracellular niche in the mucosal epithelium (between the epithelia and its basal lamina). Larvae then molt to adult worms within 2 – 5 days. After that, the adult worm mates in the non-membrane-bound portion of the enteric columnar epithelium. The fertilized females burrow deep into the cytosol of cells of the enteric mucosa and probably the absorptive and goblet cells and discharge newborn larvae in the blood circulation (5 to 15 days post-infection) (CDC, 2023).
The new generation of larvae (L1) enters the skeletal muscle cells to establish their niches, “the nurse cells,” and increase in size from 100 to 1000 μm, i.e., ten times. Literally, the infected muscle cell becomes dominated by the parasite to serve the nourishment and protection of the larvae. The development of nurse cells is associated with multiple cellular alterations and the construction of a host-derived collagen capsule. Skeletal muscles have been recognized as a unique site for the formation of nurse cells (Despommier, 1998). However, the molecular processes governing these intricate cellular alterations are largely unknown, and there are few protein markers for inducing the infected-cell phenotype (Beiting
Proposing a parasite-host interface will stimulate the development of novel anthelmintic strategies and provide a better understanding of how parasite’s strategies adjust while establishing infection at the host. For example, CDX2 expression in the intestine, a crucial transcription factor related to the caudal-related homeobox gene family with a specific expression pattern, plays a vital role in intestinal homeostasis and mucosal immunity. Physiologically, CDX2 affords the stem cells with the information that maintains the proliferation of the mucosal epithelium, the differentiation of the normal phenotype, the regulation of the cell cycle, cellular adhesion, and nutrition (Freund
In the muscles, Cyclin D1 is a physiologically critical protein that encodes the regulatory subunit of a holoenzyme that phosphorylates and deactivates the retinoblastoma protein and accelerates cell cycle progression through the G1-S phase (Mademtzoglou & Relaix, 2022). CD34 is a transmembrane sialomucin receptor biomarker and an indicator for hematopoietic progenitors and stem cells. Yet, expression of CD34 occurs in various cell types, for example, the interstitial dendritic cells, the epithelial progenitors, and the vascular endothelial progenitor cells (Alfaro
Another point is the pharmacology-host-parasite interface, where albendazole, the classic therapy, has been criticized for its poor activity against encysted
Based on the data above and hypothesis, the current study was to assess the histopathological changes in the intestine and muscle phases following infection with
One hundred Swiss albino male mice were employed in the current research. Involved animals were pathogen-free, laboratory-bred, weighing 25
The mice were divided into five major groups (Group 1 – 5), each involving 30 mice; 15 mice were determined for the intestinal phase to be sacrificed on day 7 p.i. (subgroup A), while the other 15 animals were designated for the muscular phase and were sacrificed on day 35 p.i. (subgroup B). Nevertheless, an equal number of mice (n = 10) was blindly selected per subgroup to avoid statistical bias due to the variant mortality rates to accomplish further assessments (Fig. 1).
Experimental design.
Each mouse received 250 ± 50 infectious
IVM (Iverzine 6 mg tablets, Uni Pharma, Egypt) and ACV (Acyclovir 200 mg tablets, Memphis, Egypt) were dissolved in distilled water. Referring to Paget and Barnes (1964), doses were estimated by renovating the therapeutic human doses to doses for animals. IVM was orally administered on days 1 and 5 p.i. for the intestinal phase (subgroup A) and on days 15 and 30 p.i. for the muscular phase (subgroup B) at a dose of 4 μg /mouse/day (Basyoni and El-Sabaa, 2013). Also, oral ACV was given at a dose of 10 mg/kg/day for 7 days in subgroup A (starting from day 1 p.i.) and in subgroup B (starting from day 28 p.i.) for, respectively, the intestinal and muscular phases (Quenelle
In the four groups of mice receiving the current protocol of infection and treatment, survival rate and body weight in grams (g) were detected versus their healthy control every day for the initial 7 days p.i. in subgroup A and at the end of the study period, i.e., day 35 p.i. in subgroups B.
To assess the impact of the current therapeutic protocols on adults and larvae, animals were respectively sacrificed under anesthesia by cervical dislocation 7 days p.i. for the intestinal phase (subgroups A) and 35 days p.i. for the muscular phase (subgroups B) (Pan
On day 7 p.i., the number of mature helminths per milliliter of intestinal fluid was microscopically identified. The procedure involved slicing the small intestines into small fragments after being longitudinally opened and saline. After 2 hours of incubation of the specimens in PBS at 37 °C, the adult parasites were gathered and enumerated using dissecting microscopy.
Histological assessment and quantification of adults and larvae in tissues
On days 7 and 35, after infection and therapeutic challenge, the small intestine and muscles were successively obtained from infected animals and fixed for 24 h in 4 % formalin. Then, tissues were embedded in paraffin, and cut sections of 3-μm-thickness sections were stained for histological examination using the routine H and E stain. Intestinal sections of each group were assessed for the architecture of the infected villi: villus (V) length/crypt (C) depth, counts of goblet cells/villus, counts of crypts/villus, counts of Paneth cells/crypt, and counts of inflammatory infiltrates/villus. The assessment was done in 20 villi/mouse, and mean values were calculated. The larvae, the nursing cells, and the surrounding capsule were investigated in the muscle cut sections. Average scoring of the muscular changes was done for each group in 10 larvae (randomly selected)/mouse regarding (1) larvae structures: intact-altered, (2) capsule inflammatory infiltrates: mild-moderate-intense, and (3) nurse cell: intact-altered. Adults in intestinal cut-sections were enumerated per 100 villi; also, the encapsulated larvae on muscle sections were enumerated per low power field (40×) using the gridding method (El Saftawy
For immunological staining, paraffin sections from all study groups’ small intestine and muscles were prepared on adhesive-coated glass slides. Hydrogen peroxide 3 % was used to hamper endogenous peroxide after being de-waxed and rehydrated. Slides were placed in a thermostatic bath filled with preheated ethylene diamine tetra acetic acid (EDTA) for 30 minutes at 98 °C in order to retrieve the antigens. Thereafter, cooling down was done for 20 minutes at ambient temperature. The slides containing intestinal specimens were incubated with the anti-CDX2 antibody [EPR2764Y] (#ab76541). However, muscle specimens were incubated with the monoclonal antibodies anti-Cyclin D1 antibody - C-terminal (#ab185241) and anti-CD34 antibody [EP373Y] (#ab81289). Meyer’s hematoxylin was utilized as a counterstain after the diaminobenzidine (DAB) reaction to visualize the antigen-antibody combination. In the negative control, the primary antibody was omitted. A brown stain determined the positive immune reaction; as long it is not an artifact or a background (El Saftawy
The pathological and morphometric analysis was carried out using a Leica Qwin 500 Image Analyzer (LEICA Imaging Systems Ltd., Cambridge, England). Optical density (OD) was automatically measured in ten fields, and quantitative values were saved for further statistical analysis (El Saftawy
Data were coded and entered using the statistical package for the Social Sciences (SPSS) version 28 (IBM Corp., Armonk, NY, USA). Data was summarized using mean and standard deviation for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. Comparisons between groups were done using analysis of variance (ANOVA) with multiple comparisons post hoc test in normally distributed quantitative variables while non-parametric Kruskal-Wallis test and Mann-Whitney test were used for non-normally distributed quantitative variables (Chan, 2003a). A chi-square (c2) test was performed to compare categorical data. An exact test was used when the expected frequency was less than 5 (Chan, 2003b). Correlations between quantitative variables were done using the Spearman correlation coefficient (Chan, 2003c). P-values less than 0.05 were considered statistically significant.
The study was approved by the Institutional Animal Care and Use Committee (CU-IACUC), Faculty of Sciences, Cairo University (CU: III-F-74-22). All procedures of the current experiment and study were in accordance with international guidelines for the use of laboratory animals.
The overall survival rate of mice showed insignificant differences between groups, with a p-value of 0.213. Yet, in the combined treatment (IVM + ACV), the survival rate was 93.3 %, while in the infected untreated mice, it was 73.3 %. Healthy control had a survival rate of 100 %. The mean weight in grams in all treated groups versus infected untreated animals was p-value ≤ 0.001 at either day 7 p.i. or day 35 p.i.. Further details concerning body weight are illustrated in Table 1.
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 |
11a | 73.3% | 12a,b | 80.0% | 12a,b | 80.0% | 14a,b | 93.3% | 15b | 100.0% |
g – grams; Mo – mean; in the superscripts – similar letters, p-value >0.05, while different letters p-value < 0.05.
Intestinal phase of
As shown in Table 2, the burden of adult worms in the intestinal tissues at day 7 p.i. was determined as the number of adults/ml of enteric fluid and the number of adults/100 villi. The mean adult count in infected untreated animals (subgroup1A) was estimated to be approximately 173
Counts of
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 |
No. – number; Mo – mean; R% – reduction rate; NA – not applicable, in the superscripts – similar letters, p-value > 0.05 while different letters p-value < 0.05.
The V length /C depth ratios in IVM and ACV, either as monotherapies or in combination, were restored versus infected untreated mice with p-value < 0.001. However, the V length /C depth ratio amelioration was more pronounced in the combined treatment (5.07
Regarding goblet cell counts/villus, treated groups showed p-values < 0.001 compared with infected untreated animals. Treated groups were of insignificant differences (p-value = 1). Nevertheless, only combined treatment versus healthy mice showed p-value = 1.
Counts Paneth cells/crypt and likewise the counts of crypts/villus in infected untreated mice versus all treated groups were of p-value < 0.001. Nonetheless, all therapeutic protocols were of p-value > 0.05 versus healthy control. Further details are in Table 3 and Figures 2 and 3.
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 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.
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 |
No. – number; Mo – mean; V – villus; C – crypt; in the superscripts – similar letters, p-value > 0.05, while different letters p-value < 0.05.
The mean counts of
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 |
NA – not applicable; in the superscripts – similar letters, p-value > 0.05, while different letters p-value < 0.05.
Significant differences were recorded between infected untreated mice (subgroup 1B) and treated subgroups (p-value < 0.001). Infected untreated mice showed the presence of intact larvae encompassing stichocyte, immature reproductive tract, lateral cord, intestinal tract, and the septum that separates the larvae from the nurse cell. In addition, the mature nurse cells showed eosinophilic cytoplasm and were surrounded by collagen capsules and intense inflammatory infiltrates. Notably, satellite cells under the basal lamina of the nurse cell and multiple nuclei invading the nurse cell cytoplasm were frequently present. The surrounding muscle fibers were atrophied in Fig. 4. Drug regimens altered the structure of the larvae and the nurse cells. Of note, IVM, not ACV-treated murine models, revealed scanty and relatively basophilic cytoplasm. Further details concerning histological parameters are shown in Table 5.
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.
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% |
In the superscripts, similar letters, p-value > 0.05 while different letters p-value < 0.05.
In IVM-treated mice (Fig. 6E) and ACV-treated animals (Fig. 6F), the mean OD of CDX2 was 0.58
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 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
Cyclin D1 was profoundly expressed in infected untreated animals (0.82
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.
The lowest levels of CD34 expression (0.19
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.
Overall, CDX2 in intestinal tissues (day 7 p.i.) positively correlated with the counts of lymphocytes, goblet cells, Paneth cells, crypts, and the adult and larva burden in tissue with p-values < 0.001.
Nonetheless, there were negative correlations between CDX2 in the intestine and the V length /C depth ratios (7-day p.i.) (r = −0.496, p-value < 0.001) and weight gain (days 7 and 35 p.i.) (r = −0.717 and −0.684 respectively, p-values < 0.001), (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.).
Likewise, Cyclin D1 in muscle tissue (day 35 p.i.) showed a negative correlation with V length /C depth ratios (day 7 p.i.) (r = −0.609, p-value < 0.001) and body weight (days 7 and 35 p.i.). (r = −0.853 and −0.829 respectively, p-value < 0.001). In contrast, other histological parameters in intestinal and parasite burden positively correlated (p-values < 0.001).
In contrast, the optical densities of CD34 in the muscles (day 35 p.i.) showed a positive correlation with V length /C depth ratios (day 7 p.i.) (r = 0.647, p-value < 0.001) and body weight (days 7 and 35 p.i.) (r = 0.727 and 0.746 respectively, p-value < 0.001). Yet, a negative correlation was present between the expression of the muscular CD34 and other intestinal parameters and parasite burden (p-value < 0.001), (Table 6).
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 |
No., the number of mice, 50 mice in all parameters except in parasite burden was determined on 40 mice as healthy controls (10 mice) were not involved.
Intestinal CDX2 and muscular CyclinD1 correlated positively with an r-value of 0.847 (p-value < 0.001). Muscular CD34 and intestinal CDX2 were negatively correlated (r = −0.776, p-value < 0.001), and CD34 and Cyclin D1 in muscles were likewise negatively correlated (r = −0.883, p-value < 0.001), (Table 6).
The present work showed an improved survival rate related to the three therapeutic lines of treatment (IVM, ACV, and combined treatment). IVM has been approved for the anti-helminthic therapy of onchocerciasis, strongyloidiasis, and filariasis (Nicolas
Infected untreated mice showed a significant reduction in body weight.
The combination administration of IVM and ACV monotherapies showed a significant reduction in the intestinal worm count per milliliter of enteric fluid and per 100 villi. Martin
Overall, the three therapeutic regimens showed restored V length /C depth ratios. Sarkar
Regarding IVM, Yahia
Significant increases in the goblet and Paneth cells; crypt hyperplasia was a remarkable trait in the infected untreated animals. On the contrary, the three therapeutic protocols applied in this study significantly ameliorated these histological changes. Since early literature, the expulsion of adult worms appeared to be related to the histological repair of crypt hyperplasia and villus atrophy. Yet, the complete recovery is immune-dependent, and T cell depletion in mice exhibits delayed recovery and atypical localization of the nematode (Manson-Smith
The muscle phase of infected untreated mice revealed the presence of whole and intact encapsulated larvae. The formation of nurse cells is complicated and involves molecular responses in the infected myocyte and satellite cells (Wu
Infected untreated mice showed capsule formation with intense inflammatory infiltrates that were reduced in IVM and ACV monotherapies and combined treatments. Beiting
In the intestinal phase, the immunohistochemical expression of epithelial CDX2 in infected untreated mice showed increased values that were positively correlated with parasite burden and inflammatory parameters in the intestine. Yet, the V length /C depth ratio and weight gain showed a negative correlation. Jahan
CDX2 expression in mice treated with combination treatment (IVM+ACV) and the healthy control showed insignificant differences. Likewise, in COVID-19-related studies, ACV and IVM showed efficient anti-inflammatory effects (DiNicolantonio
In the muscle phase, Cyclin D1 in infected untreated mice was over-expressed with a positive correlation to intestinal inflammation and parasite burden, negatively correlated with the V length /C depth ratio and body weight. Peer
Regarding ACV, a prior study showed that acute T-cell leukemia exerts cytotoxic action in Jurkat cells by inhibiting the cell cycle in the G1 and S phase phases and inducing cell apoptosis (Benedetti
The muscle phase also showed a reduction in the CD34 expression in the infected untreated mice with a negative correlation to intestinal inflammation and parasite burden and a positive relationship with ameliorated V length /C depth ratios and body weight. CD34 progenitor cells produce much more efficient and increased levels of Th2 cytokines than Th2 cells (Allakhverdi & Delespesse, 2012). Notably, the essential role of Type 2 immunity against the nematode is consequently necessary (Stear
CD34 expression was ameliorated following treatment and, most notably, with the combined regimen. CD34 induces motility of satellite cells (progenitor cells) and their entrance into proliferation to enable competent regeneration of skeletal muscle (Alao, 2007). CD34 was also found to recover neovascularization (Ribatti, 2007). Other cellular functions involve the adhesion of lymphocytes to the walls of blood vessels and cell morphogenesis (Sidney
Intestinal CDX2 and muscular Cyclin D 1 exhibited a positive correlation. CDX2 in chronic gastritis was speculated to promptly induce intestinal metaplasia (Eda
Expressions of CD34 and Cyclin D1 in muscles were negatively correlated. Sadr
Expression of muscular CD34 and intestinal CDX2 were negatively correlated.
Overall, the three therapeutic lines of treatment (IVM, ACV, and combined treatment) showed an improved survival rate and body weight compared with the infected untreated mice. The intestine of infected untreated mice showed higher counts of adult worms, lymphocytes, goblet cells, Paneth cells, and crypts, whereas the V length /C depth ratio recorded the lowest values. IVM and ACV monotherapies and combinations showed optimistic results regarding V length /C depth ratios and worm burden. Yet, lymphocyte counts were the only histopathological criteria that recorded lower counts in the ACV-treated groups (monotherapy and combined with IVM) compared with IVM-treated and infected untreated mice. The muscle phase of infected untreated mice revealed the definite criteria of encapsulated larvae. However, the combined treatment showed significant disruption of the encapsulated larvae and appeared to enhance muscle regeneration. The monotherapies achieved variable disruptive effects on the structure of the encapsulated larvae. In infected untreated mice, CDX2 and CyclinD1 showed a positive correlation with intestinal inflammation; nevertheless, CD34 revealed a negative correlation. CDX2 and CyclinD1 were positively correlated. CD34 was negatively correlated with CDX2 and CyclinD1. IVM +ACV significantly amended CDX2, CyclinD1, and CD34 expressions compared with monotherapies.
The current study paved the way for many perspectives, such as tumorigenicity versus the antitumor effects of