Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro

The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5–16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Expression of OLFM4 and Lysozyme During Necrotizing Enterocolitis and Volvulus in Newborns: a Comparative Observational Research Study

Background: In neonatal patients with necrotizing enterocolitis (NEC) and volvulus the inflammatory response is mediated by a plurality of different proteins. The proteins olfactomedin 4 (OLFM4) and lysozyme (LYZ) are part of the intestinal mucosal defense and especially OLFM4 has rarely been evaluated in neonatal gastrointestinal diseases. The aim of this study was to compare the expression levels of OLFM4 and lysozyme during NEC and volvulus in neonates. Methods: Intestinal tissues of patients with NEC and patients with volvulus were examined using immunohistochemical staining of OLFM4 and lysozyme of formalin-fixed and paraffin-embedded sections of resected tissue. Staining-positive tissues were semi-quantitatively scored from 0 (no staining), 1 (weak staining), 2 (moderate staining) to 3 (highly intense staining) by two individual investigators.Results: Both applied antibodies against OLFM4 showed different staining patterns with higher staining intensity of the antibody OLFM4 (D1E4M). OLFM4 (median score of the antibody OLFM4 (D1E4M): 3.0) and lysozyme (median score: 3.0) are highly expressed in intestinal and immune cells during NEC. The expression of OLFM4 and lysozyme in tissue with intestinal volvulus was also observable (median score of the antibody OLFM4 (D1E4M): 1.25) and median score of the antibody against LYZ: 2.0), but lower levels could be seen in comparison to tissue with NEC (p=0.033 and p=0.037, respectively).Conclusions: Both proteins, OLFM4 and lysozyme, may play a role in the pathogenesis of NEC and volvulus in neonatal patients, but the exact mechanisms of OLFM4 and lysozyme function and their role in immunological responses have not yet been resolved. These observations add new insights as basis for further large-scale population research.

Biallelic Mutations in WDR12 is Associated With Male Infertility With Tapered-Head Sperm

Teratozoospermia is a rare disease associated with male infertility. Unfortunately, approximately 30% of the genetic causes associated with teratozoospermia remain unknown. Several recurrent genetic mutations have been reported to be associated with globozoospermia, macrozoospermia and acephalic spermatozoa, whereas the genetic basis of tapered-head sperm is relatively less well-understood. In this study, whole-exome sequencing (WES) identified a homozygous WD repeat domain 12 (WDR12) (p.Ser162Ala/c.484T>G) variant in an infertile patient with tapered-head sperm from a consanguineous Chinese family. Bioinformatic analysis predicted this mutation to be a pathogenic variant. To further verify the effect of this variant, we analyzed WDR12 protein expression in the patient’s spermatozoa by western blot and found WDR12 to be significantly down-regulated. Also, we found that WDR12 expression is increased in pachytene spermatocytes, and intense staining was visible throughout the round spermatids in mouse testis. Based on our results, we concluded that a rare biallelic pathogenic missense variant (p.Ser162Ala/c.484T>G) in the WDR12 gene causes teratozoospermia. These results will provide novel insights into understanding the molecular mechanisms of male infertility and will help clinicians provide accurate diagnoses.

Spindle cell squamous cell carcinoma exhibiting prominent neutrophil phagocytosis: a case report

Background Spindle cell squamous cell carcinoma is an uncommon variant of squamous cell carcinoma; its diagnosis is sometimes challenging because it histopathologically resembles neoplastic or reactive spindle cell lesions of mesenchymal origins. Here, we report a rare case of spindle cell squamous cell carcinoma exhibiting prominent neutrophil phagocytosis. Case presentation A 69-year-old Japanese man presented with pain and a polypoid mass on the lower left gingiva. He had received chemoradiotherapy for squamous cell carcinoma of the buccal mucosa 15 years prior to this consultation. In addition, he was treated for mandibular osteonecrosis 6 years after chemoradiotherapy without evidence of cancer recurrence. A biopsy revealed atypical spindle or pleomorphic cells scattered in the edematous and fibrin-rich stroma; however, no malignant squamous components were apparent. These atypical cells frequently contained neutrophils within their cytoplasm that formed cell-in-cell figures. Immunohistochemically, the atypical cells were negative for cytokeratins, epithelial membrane antigen, and E-cadherin, but positive for p63, vimentin, and p53. Although these findings suggested spindle cell squamous cell carcinoma, it was difficult to reach a definitive diagnosis. Based on a clinical diagnosis of a malignant tumor, the patient underwent a hemimandibulectomy. The surgically resected specimen had a typical spindle cell squamous cell carcinoma histology consisting of biphasic spindle cells and conventional squamous cell carcinoma components. Moreover, the surgical specimen also exhibited spindle tumor cells that frequently included neutrophils, around which intense staining for lysosomal-associated membrane protein 1 and cathepsin B was observed. This suggested that the cell-in-cell figures represent active neutrophil phagocytosis by tumor cells, and not emperipolesis. Conclusion The presence of neutrophil phagocytosis may be a potent indicator of malignancy.

Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium

Epithelial wound healing is essential to repair the corneal barrier function after injury and requires coordinated epithelial sheet movement over the wounded region. The presence and role of pannexin1 on multilayered epithelial sheet migration was examined in unwounded and wounded corneal epithelium from C57BL/6J (B6) control and diet-induced obese (DiO) mice, a pretype 2 diabetic model. We hypothesize that pannexin1 is dysregulated, and the interaction of two ion-channel proteins (P2X7 and pannexin1) is altered in pretype 2 diabetic tissue. Pannexin1 was found to be present along cell borders in unwounded tissue, and no significant difference was observed between DiO and B6 control. However, an epithelial debridement induced a striking difference in pannexin1 localization. The B6 control epithelium displayed intense staining near the leading edge, which is the region where calcium mobilization was detected, whereas the staining in the DiO corneal epithelium was diffuse and lacked distinct gradation in intensity back from the leading edge. Cells distal to the wound in the DiO tissue were irregular in shape, and the morphology was similar to that of epithelium inhibited with 10Panx, a pannexin1 inhibitor. Pannexin1 inhibition reduced mobilization of calcium between cells near the leading edge, and MATLAB scripts revealed a reduction in cell-cell communication that was also detected in cultured cells. Proximity ligation was performed to determine if P2X7 and pannexin1 interaction was a necessary component of motility and communication. While there was no significant difference in the interaction in unwounded DiO and B6 control corneal epithelium, there was significantly less interaction in the wounded DiO corneas both near the wound and back from the edge. The results demonstrate that pannexin1 contributes to the healing response, and P2X7 and pannexin1 coordination may be a required component of cell-cell communication and an underlying reason for the lack of pathologic tissue migration.

Immunohistochemical expression of macrophages in chronic periapical lesions

Objective: To analyze the expression of macrophages in periapical granulomas (PGs) and radicular cysts (RCs). Methodology: We selected 264 cases of chronic periapical lesions stored at the Laboratory of Pathology, Dental School of Pernambuco (FOP)/UPE, including 89 PGs and 175 RCs. Seventy-nine cases, 23 PGs and 56 RCs, were submitted to immunohistochemical analysis by the streptavidin-biotin method using anti-CD68 antibody. The chi-square and Fisher’s exact tests were used to determine the existence of associations with categorical and clinical variables, adopting a 95% confidence level (p≤0.05). Results: We observed immunoexpression of CD68 in 83% of cases. The number of CD68+ cells (score 3 or 4) was higher in PGs, with predominantly intense staining. Significant differences in the number of CD68+ cells and staining intensity were observed between the chronic periapical lesions analyzed. Conclusion: The presence of CD68+ cells in periapical tissues may indicate the development, maintenance and/or severity of the inflammation-mediated immune response, which is more intense in PG.

Activation of Rac1-Mineralocorticoid Receptor Pathway Contributes to Renal Injury in Salt-Loaded db/db Mice

The progression of diabetic kidney disease (DKD), a leading cause of end-stage kidney disease, involves mineralocorticoid receptor (MR) activation. We previously identified crosstalk between the small guanosine triphosphatase (GTPase) RAS-related C3 botulinus toxin substrate 1 (Rac1) and MR, but the role of Rac1-MR pathway activation in the progression of DKD is not clear. We performed uninephrectomy on type 2 diabetic mouse models, db/db (UNx-high salt [HS] db/db ), and their lean control, db/m (UNx-HS db/m ), at 4-week postpartum, and fed them a high-salt diet for 10 weeks. To evaluate the involvement of the Rac1-MR pathway in the DKD progression, we investigated the effects of the nonsteroidal MR antagonist, finerenone, and the Rac1 inhibitor, NSC23766, on blood pressure and glomerular injury in UNx-HS db/db mice. UNx-HS db/db mice with hyperaldosteronism showed hypertension and hypokalemia with increased cleaved α-epithelial sodium channel expressions and massive albuminuria, accompanied by glomerular injury with nodular lesions, which is a characteristic finding in human diabetic nephropathy. Expressions of active Rac1 and serum-and glucocorticoid-induced protein kinase 1 (Sgk1), a downstream molecule of MR signaling, in the renal cortex and isolated glomeruli, significantly elevated in UNx-HS db/db mice, associated with intense staining of active Rac1 in glomerular podocytes, but both hypertension and renal injury were ameliorated by NSC23766 and finerenone, associated with Sgk1 inhibition, suggesting that Rac1-MR activation contributes to hypertension and podocyte injury. In conclusion, salt-induced activation of Rac1-MR pathway in distal tubules and glomeruli is involved in DKD progression through hypertension and glomerular injury, respectively. This finding highlights MR antagonism along with Rac1 inhibition as a novel strategy for DKD treatment.

Pulmonary Sclerosing Pneumocytoma: A Pre and Intraoperative Diagnostic Challenge. Report of Two Cases and Review of the Literature

Pulmonary sclerosing pneumocytoma is a rare benign pulmonary tumor of primitive epithelial origin. Because of the unspecific radiological features mimicking malignancies and its histological heterogeneity, the differential diagnosis with adenocarcinoma and carcinoid tumors is still challenging. We report our experience of two cases of sclerosing pneumocytoma, as well as a review of the literature. Immunohistochemical findings showed intense staining of the cuboidal epithelial cells for cytokeratin-pool and TTF-1, with focal positivity for progesterone receptors. Round and spindle cells expressed positivity for vimentin, TTF-1 and focally for the progesterone receptor. Cytologic diagnosis of pulmonary pneumocytoma requires the identification of its dual cell population, made up of abundant stromal cells and fewer surface cells. Since the pre- and intraoperative diagnosis should guide surgical decision making, obtaining a sufficient specimen size to find representative material in the cell block is of paramount importance.

CD73 expression in normal, hyperplastic, and neoplastic thyroid: a systematic evaluation revealing CD73 overexpression as a feature of papillary carcinomas

CD73 converts AMP to adenosine, an immunosuppressive metabolite that promotes tumorigenesis. This study presents a systematic evaluation of CD73 expression in benign, hyperplastic, and neoplastic thyroid. CD73 expression was assessed by immunohistochemistry in 142 thyroid samples. CD73 was expressed in normal thyroid (3/6) and goiter (5/6), with an apical pattern and mild intensity. Apical and mild CD73 expression was also present in oncocytic cell adenomas/carcinomas (9/10; 5/8) and in follicular adenomas/carcinomas (12/18; 23/27). In contrast, papillary thyroid carcinomas featured extensive and intense CD73 staining (49/50) (vs. normal thyroid/goiter, p < 0.001). Seven of nine anaplastic carcinomas were CD73-positive with heterogeneous extensiveness of staining. Medullary and poorly differentiated carcinomas were mostly CD73-negative (1/6; 2/2). These results were corroborated by NT5E mRNA profiling. Papillary carcinomas feature enhanced CD73 protein and mRNA expression with distinct and intense staining, more pronounced in the invasive fronts of the tumors.

THE ORAL SUCKER OF SOME GASTROINTESTINAL TREMATODES: HISTOLOGICAL AND HISTOCHEMICAL RESEARCH

We conducted histological and histochemical studies of the oral sucker of gastrointestinal trematodes of the family Paramphistomatidae, Fischoeder, 1901 living in the rumen of ruminants, namely, Liorchis scotiae, Paramphistomum cervi and Paramphistomum ichikawai. The oral sucker of paramphistomes and the Liorchis scotiae trematode is a complex muscular organ modified into a pharynx-sucker, the muscle complex of which is formed from longitudinal, circular and radial muscle fibers. In the thick pharynx wall, we found secretory cells, single neurosecretory cells and many desmoblastic cells of connective tissue. Histochemical stains showed intense staining with alcian blue, toluidine blue, bromophenol blue and a positive periodic acid Schiff reaction, which indicates the presence of glycosaminoglycans, total proteins and glycoproteins. Due to this structure, the pharynx sucker of gastrointestinal trematodes is involved in capturing food and evacuating its undigested residues from the helminth's body to the outside. In addition, the substances produced by secretory cells of the pharynx carry out a partial chemical treatment of the food consumed by the parasite and protect the parasite from substances that are metabolites of the host tissues and waste products of symbionts and commensals that inhabit the rumen of ruminants. Thus, trophic adaptation of the parasite in its ecological niche, the rumen of polygastric mammals, is ensured.