Infectious Keratitis: Characterization of Microbial Diversity through Species Richness and Shannon Diversity Index.

Purpose: To characterize microbial keratitis diversity utilizing species richness and Shannon Diversity Index. Methods: Corneal impression membrane was used to collect samples. All swabs were processed and analyzed by Biolab Laboratory (level V-SSN Excellence: ISO 9001:2015), Biolab Srl (Ascoli Piceno, Italy). DNA extraction, library preparation, and sequencing were performed in all samples. After sequencing, low-quality and polyclonal sequences were filtered out by the Ion software. At this point, we employed Kraken2 for microbial community analysis in keratitis samples. Nuclease-free water and all the reagents included in the experiment were used as a negative control. The primary outcome was the reduction in bacterial DNA (microbial load) at T1, expressed as a percentage of the baseline value (T0). Richness and Shannon alpha diversity metrics, along with Bray-Curtis beta diversity values, were calculated using the phyloseq package in R. Principal coordinate analysis was also conducted to interpret these metrics. Results: 19 samples were included in the study. The results exhibited a motley species richness, with the highest recorded value surpassing 800 species. Most of the samples displayed richness values ranging broadly from under 200 to around 600, indicating considerable variability in species count among the keratitis samples. Conclusions: A significant presence of both typical and atypical bacterial phyla in keratitis infections, underlining the complexity of the disease's microbial etiology.

Topical Antiseptics in Minimizing Ocular Surface Bacterial Load Before Ophthalmic Surgery: A Randomized Controlled Trial.

PURPOSE: To investigate the reduction of the ocular surface bacterial load induced by 2 commercially available ophthalmic antiseptic formulations, povidone-iodine (PVI) 0.6% and chlorhexidine (CLX) 0.02%, before ocular surgery. DESIGN: Randomized controlled trial. METHODS: Seventy adult patients undergoing intraocular surgery (phacoemulsification) were randomized to receive in the index eye PVI (group A) 4 times a day for 3 days or CLX (group B) 4 times a day for 3 days before surgery. The untreated eye was used as control. A conjunctival swab was taken in both eyes before (T0) and after (T1) therapy. Microbial DNA was quantified with real-time polymerase chain reaction (PCR) analysis. The Mick algorithm was used to compare the abundance of each genus/genera against the distribution of abundances from the reference. At T1, patients filled a questionnaire to evaluate therapy-induced symptoms. Primary outcome was the reduction of bacterial DNA at T1 (microbial load), vs control arm, expressed as mean number of real-time PCR cycle times (CTs). Secondary outcomes were taxonomic composition, differential abundance, and therapy-induced ocular symptoms. RESULTS: The T0-T1 difference in CT was significant in group B, but not in group A (mean [95% CI], 0.99 [0.33] vs 0.26 [0.15], P < .001, and 0.65 [0.3] vs 0.45 [0.41], P = .09, respectively). The taxonomic composition, alpha, and beta diversity remained consistent at all time points in both groups. The rate of patients reporting therapy-induced ocular symptoms and the mean discomfort grade were greater in group A than in group B (97% vs 26% and 4.97±2.48 vs 0.66±1.53, respectively). CONCLUSIONS: Compared with PVI 0.6%, CLX 0.02% induced a greater reduction of ocular surface bacterial load, with no significant alterations of the taxonomic composition. Moreover, CLX was better tolerated than PVI.

Ocular Surface Microbiota in Naïve Keratoconus: A Multicenter Validation Study.

In the field of Ophthalmology, the mNGS 16S rRNA sequencing method of studying the microbiota and ocular microbiome is gaining more and more weight in the scientific community. This study aims to characterize the ocular microbiota of patients diagnosed with keratoconus who have not undergone any prior surgical treatment using the mNGS 16S rRNA sequencing method. Samples of naïve keratoconus patients were collected with an eNAT with 1 mL of Liquid Amies Medium (Copan Brescia, Italy), and DNA was extracted and analyzed with 16S NGS. The microbiota analysis showed a relative abundance of microorganisms at the phylum level in each sample collected from 38 patients with KC and 167 healthy controls. A comparison between healthy control and keratoconus samples identified two genera unique to keratoconus, Pelomonas and Ralstonia. Our findings suggest that alterations in the microbiota may play a role in the complex scenario of KC development.

Tailoring DNA Vaccines: Designing Strategies Against HER2-Positive Cancers.

The crucial role of HER2 in epithelial transformation and its selective overexpression on cancer tissues makes it an ideal target for cancer immunotherapies such as passive immunotherapy with Trastuzumab. There are, however, a number of concerns regarding the use of monoclonal antibodies which include resistance, repeated treatments, considerable costs, and side effects that make active immunotherapies against HER2 desirable alternative approaches. The efficacy of anti-HER2 DNA vaccination has been widely demonstrated in transgenic cancer-prone mice, which recapitulate several features of human breast cancers. Nonetheless, the rational design of a cancer vaccine able to trigger a long-lasting immunity, and thus prevent tumor recurrence in patients, would require the understanding of how tolerance and immunosuppression regulate antitumor immune responses and, at the same time, the identification of the most immunogenic portions of the target protein. We herein retrace the findings that led to our most promising DNA vaccines that, by encoding human/rat chimeric forms of HER2, are able to circumvent peripheral tolerance. Preclinical data obtained with these chimeric DNA vaccines have provided the rationale for their use in an ongoing Phase I clinical trial (EudraCT 2011-001104-34).

Identification of relevant conformational epitopes on the HER2 oncoprotein by using Large Fragment Phage Display (LFPD).

We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines.

The human splice variant Δ16HER2 induces rapid tumor onset in a reporter transgenic mouse.

Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform "per se" mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein.

Mesenchymal/stromal gene expression signature relates to basal-like breast cancers, identifies bone metastasis and predicts resistance to therapies.

BACKGROUND: Mounting clinical and experimental evidence suggests that the shift of carcinomas towards a mesenchymal phenotype is a common paradigm for both resistance to therapy and tumor recurrence. However, the mesenchymalization of carcinomas has not yet entered clinical practice as a crucial diagnostic paradigm. METHODOLOGY/PRINCIPAL FINDINGS: By integrating in silico and in vitro studies with our epithelial and mesenchymal tumor models, we compare herein crucial molecular pathways of previously described carcinoma-derived mesenchymal tumor cells (A17) with that of both carcinomas and other mesenchymal phenotypes, such as mesenchymal stem cells (MSCs), breast stroma, and various types of sarcomas. We identified three mesenchymal/stromal-signatures which A17 cells shares with MSCs and breast stroma. By using a recently developed computational approach with publicly available microarray data, we show that these signatures: 1) significantly relates to basal-like breast cancer subtypes; 2) significantly relates to bone metastasis; 3) are up-regulated after hormonal treatment; 4) predict resistance to neoadjuvant therapies. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that mesenchymalization is an intrinsic property of the most aggressive tumors and it relates to therapy resistance as well as bone metastasis.