First microscopic, pathological, epidemiological, and molecular investigation of Leucocytozoon (Apicomplexa: Haemosporida) parasites in Egyptian pigeons.

Introduction: Leucocytozoon is an intracellular blood parasite that affects various bird species globally and is transmitted by blackfly vectors. This parasite is responsible for leucocytozoonosis, a disease that results in significant economic losses due to reduced meat and egg production. There is limited knowledge about the epidemiological pattern of leucocytozoonosis and its causative species in Egypt, particularly in pigeons. Methods: The current study involved the collection of 203 blood samples from domestic pigeons from various household breeders and local markets across Qena Province, Upper Egypt. Samples were initially examined for potential Leucocytozoon infection using blood smears, followed by an evaluation of associated risk factors. Molecular identification of the parasite in selected samples (n = 11), which had initially tested positive via blood smears, was further refined through nested PCR and sequence analysis of the mitochondrial cytochrome b gene to ascertain the Leucocytozoon species present. Additionally, histopathological examination of the liver, spleen, and pancreas was conducted on animals that tested positive by blood smears. Results: Interestingly, 26 out of 203 samples (12.08%) had confirmed Leucocytozoon infections based on microscopic analysis. Additionally, all 11 samples that initially tested positive via blood smears were confirmed positive through nested PCR analysis, and their sequencing revealed the presence of Leucocytozoon sabrazesi, marking the first report of this parasite in Egypt. The study into potential risk factors unveiled the prevalence of Leucocytozoon spp. seems host gender-dependent, with males exhibiting a significantly higher infection rate (33.33%). Additionally, adult birds demonstrated a significantly higher infection prevalence than squabs, suggesting an age-dependent trend in prevalence. Seasonality played a significant role, with the highest occurrence observed during summer (37.25%). Histopathological examination revealed the presence of numerous megaloschizonts accompanied by lymphocytic infiltration and multiple focal areas of ischemic necrosis. Conclusion: To our knowledge, this is the first study to shed light on the epidemiological characteristics and molecular characterization of leucocytozoonosis in pigeons in Egypt. Further research endeavors are warranted to curb the resurgence of Leucocytozoon parasites in other avian species across Egypt, thereby refining the epidemiological understanding of the disease for more effective control and prevention measures.

Epidemiological perspective associated with principal risk factors of Trichinella spiralis infection in pigs and humans in Egypt.

Background and Aim: In Egypt, there is a scarcity of recent data on trichinellosis in pigs and humans. Therefore, this study aimed to determine the epidemiological profile and risk factors associated with Trichinella spiralis infection as well as to assess the effectiveness of the trichinoscope and digestion technique in diagnosing trichinellosis. Materials and Methods: Data were collected on 33812 pigs slaughtered during a year at the Al-Basateen abattoir, Cairo Governorate, Egypt. The slaughtered pigs had already been examined by trichinoscope in the abattoir. The diagnostic effectiveness technique was randomly conducted on 170 pork muscle samples, which were examined using the digestion technique. Furthermore, 90 serum samples from high-risk individuals in Qena and Sohag Governorates, Upper Egypt, were analyzed using an enzyme-linked immunosorbent assay. Results: The investigation revealed that the overall prevalence was 1.06% in pigs by trichinoscope. Of the examined 170 samples, 2.35% and 3.35% were found to harbor Trichinella by trichinoscope and artificial digestion, respectively. Trichinella was identified as T. spiralis using a polymerase chain reaction (PCR) technique. A significant relationship was affirmed between the prevalence of trichinellosis and the sex and age of the examined pigs. Likewise, for the first time, there was a considerable seasonal trend in the prevalence of Trichinella with the maximum infection, which was observed during Autumn (1.18%). The prevalence of trichinellosis in humans was 10%, with a significant association with age. Conclusion: Our findings are intended to serve as a starting point for developing effective preventive and control measures for trichinellosis (as application of Hazard Analysis Critical Control Points (HACCP) in pig farms, stop feeding pigs on garbage as well as, preventing illegal slaughter of pigs outside the slaughterhouses). It also fortifies the establishment of the digestion technique because of its high specificity and sensitivity, although it is difficult to apply to a large number of samples.

Therapeutic modality of induced uterine leiomyoma with shock waves in rats: The uterine blood flow, circulating ovarian hormones and histopathological findings.

Uterine leiomyoma is the most common benign pelvic tumor and the primary indication for hysterectomy. We hypothesized tumor softening and shrinking through shock waves mechanobiological influence on fibroblasts of the induced leiomyoma in rats. Three rats served as control from thirty-three female Wistar rats subjected to leiomyoma induction using mono-sodium glutamate and estradiol benzoate. After assessing uterine leiomyoma development with Doppler ultrasonography, blood and tissue samples were collected for hormonal and histopathological analysis. Of the fifteen rats treated with shock waves, five rats were sacrificed after receiving two sessions (2S), another five rats were sacrificed after receiving four sessions (4S), and the last five rats were sacrificed after two weeks recovery period (recovered 4S). From the fifteen non-treated leiomyoma group, five rats were sacrificed after Doppler ultrasound assessment (Leiomyoma), another five rats were sacrificed with the 4S group (Leiomyoma 1Wk recovery), and the last five rats were sacrificed with the recovered 4S group (Recovered leiomyoma). The collected blood samples, estradiol (E2), Estrogen receptor, progesterone (P4), and progesterone receptor (PGR), were assayed. Total cholesterol, protein, albumin, and globulin were measured. Uterine arteries' blood flow velocities, indices, and volume were obtained. Tissue samples were stained with smooth muscle actin (SMA), trichrome-three, and (hematoxylin and eosin). Rats developed leiomyoma had the highest (P = 0.0001) gross and sonographic uterine horns diameters, uterine weight, uterine coefficient, E2, and ER. Both trichrome-three and SMA staining confirmed the leiomyoma development and the response to shock waves treatment. In conclusion, low-intensity shock waves proved curative to the induced leiomyoma.

Coculturing of mesenchymal stem cells of different sources improved regenerative capability of osteochondral defect in the mature rabbit: An in vivo study.

PURPOSE: Choosing a therapeutic cell source for osteochondral repair remains a challenge. The present study investigated coculturing mesenchymal stem cells (MSCs) from different sources to provide an improved therapeutic cell option for osteochondral repair. METHODS: Dutch and Japanese white rabbits were used in this study, the first for isolating MSCs and the second for creating an osteochondral model in the medial femoral condyle. The 26 rabbit knees were divided randomly into four groups: control ( n = 6), bone marrow-derived MSCs (BMSCs) ( n = 7), synovial tissue MSCs (SMSCs) ( n = 7), and cocultured MSCs ( n = 6). Tissue repair was assessed using the Fortier scale, and colony-forming assay was performed. RESULTS: At different cell densities, cocultured and SMSCs formed larger colonies than BMSCs, indicating their high proliferative potential. After 2 months, complete filling of the defect with smooth surface regularity was detected in the cocultured MSC group, although there was no significant difference among the therapeutic groups macroscopically. Also, tissue repair was histologically better in the cocultured MSC group than in the control and SMSC groups, due to repair of the subchondral bone and coverage with hyaline cartilage. Additionally, toluidine blue and collagen-II staining intensity in the repaired tissue was better in the cocultured MSC group than in the remaining groups. CONCLUSION: Our results suggest that cocultured MSCs are a suitable option for the regeneration capability of osteochondral defects due to their enhanced osteochondrogenic potential.

Utility of Survivin, BAP1, and Ki-67 immunohistochemistry in distinguishing epithelioid mesothelioma from reactive mesothelial hyperplasia.

Histological distinction between epithelioid mesothelioma (EM) and reactive mesothelial hyperplasia (RMH) can be challenging. The aim of this study was to assess the diagnostic utility of Survivin, Ki-67, and loss of BRCA1-associated protein 1 (BAP1) expressions in distinguishing EM from RMH using immunohistochemistry. Formalin-fixed, paraffin-embedded specimens from 78 cases of EM and 80 cases of RMH were immunohistochemically examined for Survivin, BAP1, and Ki-67. In addition, receiver operating characteristic curve analyses were performed to establish the cut-off values for Survivin and Ki-67 labelling indices. Survivin (cut-off value: 5%) had 67.7% sensitivity and 100% specificity, while Ki-67 (cut-off value: 10%) had 85.1% sensitivity and 87.5% specificity, and BAP1 had 66.2% sensitivity and 100% specificity for the differentiation of EM from RMH. Among the combinations of two markers, the combination of Survivin and BAP1 (Survivin-positive and/or BAP1-loss finding) had the highest diagnostic accuracy (sensitivity: 89.8%; specificity: 100%; accuracy: 95.3%). We recommend using the combination of Survivin and BAP1 to distinguish EM from RMH.

MUC4 immunohistochemistry is useful in distinguishing epithelioid mesothelioma from adenocarcinoma and squamous cell carcinoma of the lung.

The differential diagnosis of epithelioid mesothelioma from lung adenocarcinoma and squamous cell carcinoma requires the positive and negative immunohistochemical markers of mesothelioma. The IMIG guideline has suggested the use of Calretinin, D2-40, WT1, and CK5/6 as mesothelial markers, TTF-1, Napsin-A, Claudin 4, CEA as lung adenocarcinoma markers p40, p63, CK5/6, MOC-31 as squamous cell markers. However, use of other immunohistochemical markers is still necessary. We evaluated 65 epithelioid mesotheliomas, 60 adenocarcinomas, and 57 squamous cell carcinomas of the lung for MUC4 expression by immunohistochemistry and compared with the previously known immunohistochemical markers. MUC4 expression was not found in any of 65 cases of epithelioid mesothelioma. In contrast, MUC4 expression was observed in 50/60(83.3%) cases of lung adenocarcinoma and 50/56(89.3%) cases of lung squamous cell carcinoma. The negative MUC4 expression showed 100% sensitivity, 86.2% specificity and accuracy rate of 91.2% to differentiate epithelioid mesothelioma from lung carcinoma. The sensitivity, specificity, and accuracy of MUC4 are comparable to that of previously known markers of lung adenocarcinoma and squamous cell carcinoma, namely CEA, Claudin 4 and better than that of MOC-31. In conclusion, MUC4 immunohistochemistry is useful for differentiation of epithelioid mesothelioma from lung carcinoma, either adenocarcinoma or squamous cell carcinoma.

PIM1 knockdown inhibits cell proliferation and invasion of mesothelioma cells.

Malignant mesothelioma is a major asbestos-related cancer with prolonged time lapse from the first exposure of asbestos to the development of mesothelioma. Most of mesothelioma patients show very poor prognosis, thus, an urgent improvement of its treatment is required by development of novel therapeutic strategies. RNA interference (RNAi) is a powerful tool in post-genomic research and cancer therapy through inhibition of gene expression. In the present study, we analyzed the function of PIM1 on mesothelioma cell lines with its knockdown by siRNA transfection. Here, we report that the downregulation of PIM1 led to suppression of cell proliferation by cell cycle arrest at G1 phase and suppression of cell invasion and migration. Considering the mesothelioma as rapidly growing invasive cancer, downregulation of PIM1 may have a potential role for therapeutic management of malignant mesothelioma.

MUC4, a novel immunohistochemical marker identified by gene expression profiling, differentiates pleural sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

Sarcomatoid mesothelioma, a histological subtype of malignant pleural mesothelioma, is a very aggressive tumor with a poor prognosis. Histological diagnosis of sarcomatoid mesothelioma largely depends on the histomorphological feature of spindled tumor cells with immunohistochemical reactivity to cytokeratins. Diagnosis also requires clinico-radiological and/or macroscopic evidence of an extrapulmonary location to differentiate it from lung sarcomatoid carcinoma. Although there are promising immunohistochemical antibody panels to differentiate mesothelioma from lung carcinoma, a consensus on the immunohistochemical markers that distinguish sarcomatoid mesothelioma from lung sarcomatoid carcinoma has not been reached and requires further study. We performed whole gene expression analysis of formalin-fixed paraffin-embedded tissue from sarcomatoid mesothelioma and lung sarcomatoid carcinoma and observed significant differences in the expression of MUC4 and other genes between sarcomatoid mesothelioma and lung sarcomatoid carcinoma. Immunohistochemistry demonstrated that MUC4 was expressed in the spindled tumor cells of lung sarcomatoid carcinoma (21/29, 72%) but was not expressed in any sarcomatoid mesothelioma (0/31, 0%). To differentiate sarcomatoid mesothelioma from lung sarcomatoid carcinoma, negative MUC4 expression showed 100% sensitivity and 72% specificity and accuracy rate of 87%, which is higher than immunohistochemical markers such as calretinin, D2-40 and Claudin-4. Therefore, we recommend to include MUC4 as a novel and useful negative immunohistochemical marker for differentiating sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

Use of Anti-Noxa Antibody for Differential Diagnosis between Epithelioid Mesothelioma and Reactive Mesothelial Hyperplasia.

OBJECTIVES: The histological differential diagnosis between epithelioid mesothelioma (EM) and reactive mesothelial hyperplasia (RMH) is not always straightforward. The aim of the present study was to search for new immunohistochemical markers to distinguish EM from RMH. METHODS: We evaluated and compared the expression of apoptosis-related genes in EM and RMH by real-time RT-PCR array analysis followed by clustering of significant gene expression. Immunohistochemical staining and statistical analysis of Noxa expression in 81 cases of EM and 55 cases of RMH were performed and compared with the utility of other previously reported antibodies such as Desmin, EMA, GLUT-1, IMP-3 and CD146. RESULTS: Noxa mRNA expression levels were found to be increased in EM when compared to RMH by RT-PCR array analysis. In the immunohistochemical analysis, Noxa showed sensitivity of 69.0%, specificity of 93.6% and positive predictive value of 93.0% as a positive marker of EM in distinguishing it from RMH, and these values were almost similar to IMP-3. CONCLUSION: Noxa is a marker with relatively high specificity, and can be used to distinguish EM from RMH. It would be a valuable addition to the current antibody panel used for the differential diagnosis of EM and RMH.

Differential microRNA expression profiling of mesothelioma and expression analysis of miR-1 and miR-214 in mesothelioma.

Malignant mesothelioma is a highly aggressive cancer with poor prognosis and refractory to currently available therapies. Most of the patients with advanced invasive nature are not amenable to surgical resection and/or available anticancer therapy, thus development of novel effective therapeutic regimes is needed. Aberrant expression of microRNAs (miRNAs) has been proposed to contribute to carcinogenesis and aggressiveness of mesothelioma. We analyzed miRNA expression in mesothelioma cell lines using TaqMan miRNA expression array and found significant number of miRNAs, which showed increased or lost expression. We validated the increased expression of miR-182, and miR-183 in mesothelioma cell lines by individual miRNA assays and SmartFlare miRNA probes. We further investigated the miR-1, and miR-214, which were not expressed in mesothelioma cells by real-time RT-PCR. Transfection of mesothelioma cells, ACC-Meso-1 and CRL5915, with miRNA mimic (hsa-miR-1 mimic and hsa-miR-214 mimic) led to inhibition of cell growth, invasion and migration. We paid attention to PIM1, the target gene of both miR-1 and miR-214 miRNAs and which was found overexpressed in mesothelioma cells, and miR-1 and miR-214 mimic transfection of mesothelioma cell lines showed downregulation of PIM1 by western blot analysis. The miRNAs, miR-1 and miR-214, may play a role in carcinogenesis of mesothelioma thus might be considered as candidate therapeutic targets in mesothelioma.