Hernandes F. Carvalho

Second harmonic generation microscopy as a powerful diagnostic imaging modality for human ovarian cancer

In this study we showed that second-harmonic generation (SHG) microscopy combined with precise methods for images evaluation can be used to detect structural changes in the human ovarian stroma. Using a set of scoring methods (alignment of collagen fibers, anisotropy, and correlation), we found significant differences in the distribution and organization of collagen fibers in the stroma component of serous, mucinous, endometrioid and mixed ovarian tumors as compared with normal ovary tissue. This methodology was capable to differentiate between cancerous and healthy tissue, with clear cut distinction between normal, benign, borderline, and malignant tumors of serous type. Our results indicated that the combination of different image-analysis approaches presented here represent a powerful tool to investigate collagen organization and extracellular matrix remodeling in ovarian tumors.

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes

Molecular mechanisms responsible for periodontal disease (PD) and its worsening in type 1 Diabetes Mellitus (DM1) remain unknown. Cytokine profile and expression levels of collagenases, Mmp14, and tissue inhibitors were determined, as were the numbers of neutrophils and macrophages in combined streptozotocin‐induced DM1 and ligature‐induced PD models. Increased IL‐23 (80‐fold) and Mmp8 expression (25‐fold) was found in DM1. Ligature resulted in an IL‐1β/IL‐6 profile, increased expression of Mmp8, Mmp13, and Mmp14 (but not Mmp1), and transient expression of Timp1 and Reck in non‐diabetics. PD in DM1 involved IL‐1β (but not IL‐6) and IL‐23/IL‐17, reduced IL‐6 and IL‐10, sustained Mmp8 and Mmp14, increased Mmp13 and reduced Reck expression in association with 20‐fold higher counts of neutrophils and macrophages. IL‐23 and Mmp8 expression are hallmarks of DM1. In association with the IL‐1/IL‐6 (Th1) response in PD, one found a secondary IL‐17 (Th17) pathway in non‐diabetic rats. Low IL‐6/TNF‐α suggest that the Th1 response was compromised in DM1, while IL‐17 indicates a prevalence of the Th17 pathway, resulting in high neutrophil recruitment. Mmp8, Mmp13, and Mmp14 expression seems important in the tissue destruction during PD in DM1. PD‐associated IL‐1/IL‐6 (Th1), IL‐10, and Reck expression are associated with the acute‐to‐chronic inflammation transition, which is lost in DM1. In conclusion, IL‐23/IL‐17 are associated with the PD progression in DM1. J. Cell. Physiol. 227: 2441–2450, 2012.

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

Background Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. Methodology/Principal Findings We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. Conclusions/Significance NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.

The Severity of Osteogenesis Imperfecta and Type I Collagen Pattern in Human Skin as Determined by Nonlinear Microscopy: Proof of Principle of a Diagnostic Method

Background The confirmatory diagnosis of Osteogenesis Imperfecta (OI) requires invasive, commonly bone biopsy, time consuming and destructive methods. This paper proposes an alternative method using a combination of two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) microscopies from easily obtained human skin biopsies. We show that this method can distinguish subtypes of human OI. Methodology/Principal Findings Different aspects of collagen microstructure of skin fresh biopsies and standard H&E-stained sections of normal and OI patients (mild and severe forms) were distinguished by TPEF and SHG images. Moreover, important differences between subtypes of OI were identified using different methods of quantification such as collagen density, ratio between collagen and elastic tissue, and gray-level co-occurrence matrix (GLCM) image-pattern analysis. Collagen density was lower in OI dermis, while the SHG/autofluorescence index of the dermis was significantly higher in OI as compared to that of the normal skin. We also showed that the energy value of GLCM texture analysis is useful to discriminate mild from severe OI and from normal skin. Conclusions/Significance This work demonstrated that nonlinear microscopy techniques in combination with image-analysis approaches represent a powerful tool to investigate the collagen organization in skin dermis in patients with OI and has the potential to distinguish the different types of OI. The procedure outlined in this paper requires a skin biopsy, which is almost painless as compared to the bone biopsy commonly used in conventional methods. The data presented here complement existing clinical diagnostic techniques and can be used as a diagnostic procedure to confirm the disease, evaluate its severity and treatment efficacy.

Laminin-111 and the Level of Nuclear Actin Regulate Epithelial Quiescence via Exportin-6

Nuclear actin (N-actin) is known to participate in the regulation of gene expression. We showed previously that N-actin levels mediate the growth and quiescence of mouse epithelial cells in response to laminin-111 (LN1), a component of the mammary basement membrane (BM). We know that BM is defective in malignant cells, and we show here thatxa0it is the LN1/N-actin pathway that is aberrant inxa0human breast cancer cells, leading to continuous growth. Photobleaching assays revealed that N-actin exit in nonmalignant cells begins as early as 30xa0minxa0after LN1 treatment. LN1 attenuates the PI3K pathway leading to upregulation of exportin-6 (XPO6) activity and shuttles actin out of the nucleus. Silencing XPO6 prevents quiescence. Malignant cells are impervious to LN1 signaling. These results shed light on the crucial role of LN1 in quiescence and differentiation and how defects in the LN1/PI3K/XPO6/N-actin axis explain the loss of tissue homeostasis and growth control that contributes to malignant progression.

Measurement of the hydrodynamic radius of quantum dots by fluorescence correlation spectroscopy excluding blinking.

One of the most important properties of quantum dots (QDs) is their size. Their size will determine optical properties and in a colloidal medium their range of interaction. The most common techniques used to measure QD size are transmission electron microscopy (TEM) and X-ray diffraction. However, these techniques demand the sample to be dried and under a vacuum. This way any hydrodynamic information is excluded and the preparation process may alter even the size of the QDs. Fluorescence correlation spectroscopy (FCS) is an optical technique with single molecule sensitivity capable of extracting the hydrodynamic radius (HR) of the QDs. The main drawback of FCS is the blinking phenomenon that alters the correlation function implicating in a QD apparent size smaller than it really is. In this work, we developed a method to exclude blinking of the FCS and measured the HR of colloidal QDs. We compared our results with TEM images, and the HR obtained by FCS is higher than the radius measured by TEM. We attribute this difference to the cap layer of the QD that cannot be seen in the TEM images.

Use of the second harmonic generation microscopy to evaluate chondrogenic differentiation of mesenchymal stem cells for cartilage repair

Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG) microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks, the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based cell therapy for cartilage repair.

Multimodal and non-linear optical microscopy applications in reproductive biology.

A plethora of optical techniques is currently available to obtain non‐destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four‐dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567–582, 2016.

Collagen analysis by second-harmonic generation microscopy predicts outcome of luminal breast cancer

Second-harmonic generation microscopy represents an important tool to evaluate extracellular matrix collagen structure, which undergoes changes during cancer progression. Thus, it is potentially relevant to assess breast cancer development. We propose the use of second-harmonic generation images of tumor stroma selected on hematoxylin and eosin–stained slides to evaluate the prognostic value of collagen fibers analyses in peri and intratumoral areas in patients diagnosed with invasive ductal breast carcinoma. Quantitative analyses of collagen parameters were performed using ImageJ software. These parameters presented significantly higher values in peri than in intratumoral areas. Higher intratumoral collagen uniformity was associated with high pathological stages and with the presence of axillary lymph node metastasis. In patients with immunohistochemistry-based luminal subtype, higher intratumoral collagen uniformity and quantity were independently associated with poorer relapse-free and overall survival, respectively. A multivariate response recursive partitioning model determined 12.857 and 11.894 as the best cut-offs for intratumoral collagen quantity and uniformity, respectively. These values have shown high sensitivity and specificity to differentiate distinct outcomes. Values of intratumoral collagen quantity and uniformity exceeding the cut-offs were strongly associated with poorer relapse-free and overall survival. Our findings support a promising prognostic value of quantitative evaluation of intratumoral collagen by second-harmonic generation imaging mainly in the luminal subtype breast cancer.

The origin of prostate gland-secreted IgA and IgG

The prostate secretes immunoglobulin (Ig) A (IgA) and IgG; however, how immunoglobulins reach the secretion, where the plasma cells are located, whether immunoglobulins are antigen-specific and where activation of the adaptive response occurs are still unknown. Immune cells, including CD45RA+ cells, were scattered in the stroma and not organized mucosae-associated lymphoid-tissue. IgA (but not IgG) immunostaining identified stromal plasma cells and epithelial cells in non-immunized rats. Injected tetramethylrhodamine-IgA transcytosed the epithelium along with polymeric immunoglobulin receptor. Oral immunization with ovalbumin/mesopourous SBA-15 silica adjuvant resulted in more stromal CD45RA+/IgA+ cells, increased content of ovalbumin-specific IgA and IgG, and the appearance of intraepithelial CD45RA+/IgG+ cells. An increased number of dendritic cells that cooperate in other sites with transient immunocompetent lymphocytes, and the higher levels of interleukin-1β, interferon-γ and transforming growth factor-β, explain the levels of specific antibodies. Nasal immunization produced similar results except for the increase in dendritic cells. This immunomodulatory strategy seems useful to boost immunity against genitourinary infections and, perhaps, cancer.