A dry powder formulation for peripheral lung delivery and absorption of an anti-SARS-CoV-2 ACE2 decoy polypeptide.

One of the strategies proposed for the neutralization of SARS-CoV-2 has been to synthetize small proteins able to act as a decoy towards the virus spike protein, preventing it from entering the host cells. In this work, the incorporation of one of these proteins, LCB1, within a spray-dried formulation for inhalation was investigated. A design of experiments approach was applied to investigate the optimal condition for the manufacturing of an inhalable powder. The lead formulation, containing 6% w/w of LCB1 as well as trehalose and L-leucine as excipients, preserved the physical stability of the protein and its ability to neutralize the virus. In addition, the powder had a fine particle fraction of 58.6% and a very high extra-fine particle fraction (31.3%) which could allow a peripheral deposition in the lung. The in vivo administration of the LCB1 inhalation powder showed no significant difference in the pharmacokinetic from the liquid formulation, indicating the rapid dissolution of the microparticles and the protein capability to translocate into the plasma. Moreover, LCB1 in plasma samples still maintained the ability to neutralize the virus. In conclusion, the optimized spray drying conditions allowed to obtain an inhalation powder able to preserve the protein biological activity, rendering it suitable for a systemic prevention of the viral infection via pulmonary administration.

A new experimental model to allow use of clinical-scale endoscopes in small-animal tumor models.

BACKGROUND: The evaluation of novel endoscopes may require testing in experimental tumor models, particularly when employing new biomarkers. Tumor models, however, exist almost exclusively in small animals. Therefore, we aimed to develop an experimental setting that allows the use of clinical-scale endoscopes in small animals. METHODS: In our approach, the proximal large bowel with intact blood supply is exposed on a movable and height-adjustable table. The endoscope's tip may be inserted into the bowel; the dark environment of the bowel lumen in vivo is simulated by mounting a light-tight curtain around the endoscope. Proof-of-principle experiments were done in Wag/Rij rats following cecal injection of the cell line R1H. RESULTS: Using high-definition television white-light endoscopy, narrow-band and autofluorescence imaging, and miniprobe-based confocal laser microscopy (CLM) marked differences were observed between normal mucosa and tumors. Depending on the techniques, mean examination times ranged from 3 to 10 minutes. Even after 90 minutes the colon displayed an intact blood supply, imaged by Evans blue injection and by CLM. CONCLUSION: These experiments demonstrate that our model allows in vivo examination of small-animals by clinical-scale endoscopes. Therefore, it may be useful for evaluation, at various stages of GI carcinogenesis, of both new biomarkers and endoscopic technologies.

Selected pro-inflammatory factor transcripts in bovine endometrial epithelial cells are regulated during the oestrous cycle and elevated in case of subclinical or clinical endometritis.

Endometrial cells take part in embryo-maternal communication, as well as supporting the immune system in defending against invading pathogens. The aim of the present study was to examine the mRNA expression of factors that have been suggested to be involved in both events in the bovine endometrial epithelium, namely bovine granulocyte chemotactic protein 2 (CXCL5), interleukin-1 beta (IL1B), IL6, IL8, tumour necrosis factor (TNF), cyclooxygenase 2 (PTGS2) and haptoglobin (HP). Samples were collected in vivo from cows on Days 21-27 postpartum by the cytobrush method to evaluate the correlation between inflammatory factors and uterine health (cows with signs of clinical or subclinical endometritis and healthy cows). Bovine uteri were collected at the abattoir to investigate oestrous cycle-dependent mRNA expression patterns. Real-time reverse transcription-polymerase chain reaction revealed that the expression of CXCL5, IL1B, IL8 and TNF mRNA was significantly higher in cows with subclinical or clinical endometritis compared with healthy cows. The expression of CXCL5, IL1B and IL8 mRNA was increased around ovulation compared with the luteal phase. There was no indication of either oestrous cycle-dependent expression or a correlation with uterine health for IL6, PTGS2 and HP transcripts. These results suggest that CXCL5, IL1B, IL8 and TNF may represent potential marker genes for the detection of cows with subclinical endometritis and for monitoring new therapeutic approaches.

Differential expression of cyclooxygenase 1 and cyclooxygenase 2 in the bovine oviduct.

The aim of the present study was to investigate the enzymes for the local prostaglandin (PG) biosynthesis present in the bovine oviduct during the estrous cycle to influence early reproductive events. Bovine oviducts were classified into four phases: pre-ovulatory, post-ovulatory, early-to-mid luteal, and late luteal phase, subdivided further into ipsi- or contralateral site and separated into ampulla or isthmus. Oviductal cells were gained by flushing the oviductal regions. Quantitative real-time reverse transcriptase-PCR was performed for the secretory and cytosolic phospholipases A(2) (sPLA(2)IB, cPLA(2)alpha, and cPLA(2)beta) and cyclooxygenases (COX-1 and COX-2) as the first step enzymes of PG synthesis. COX-1 and cPLA(2)beta showed significant highest mRNA expression around and before ovulation compared with the luteal phase respectively. sPLA(2)IB and cPLA(2)alpha mRNA expression was unregulated during the estrous cycle. Regional differences in mRNA content were found for sPLA(2)IB with higher mRNA expression in the ampulla than in the isthmus. Western blot analysis revealed the highest COX-1 protein content in the early-to-mid luteal phase. Immunohistochemistry demonstrated that COX-1 was localized in epithelial and smooth muscle cells, whereas COX-2 was only localized in epithelial cells. COX-2 showed a differential distribution within the epithelial cell layer suggesting a regulation on a cellular level, although the COX-2 mRNA and protein amounts did not vary throughout the estrous cycle. A COX activity assay of oviductal cells revealed that COX activity originated predominantly from COX-1 than from COX-2. Treatment of primary oviductal cells with 10 pg/ml 17beta-estradiol or 10 ng/ml progesterone resulted in a higher expression of COX-2 and cPLA(2)alpha, but not of the other enzymes. The expression pattern of these enzymes suggests that an estrous-cycle dependent and region-specific PG synthesis in the bovine oviduct may be required for a successful reproduction.