Emerging Lab-on-a-Chip Approaches for Liquid Biopsy in Lung Cancer: Status in CTCs and ctDNA Research and Clinical Validation.

Despite the intensive efforts dedicated to cancer diagnosis and treatment, lung cancer (LCa) remains the leading cause of cancer-related mortality, worldwide. The poor survival rate among lung cancer patients commonly results from diagnosis at late-stage, limitations in characterizing tumor heterogeneity and the lack of non-invasive tools for detection of residual disease and early recurrence. Henceforth, research on liquid biopsies has been increasingly devoted to overcoming these major limitations and improving management of LCa patients. Liquid biopsy is an emerging field that has evolved significantly in recent years due its minimally invasive nature and potential to assess various disease biomarkers. Several strategies for characterization of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been developed. With the aim of standardizing diagnostic and follow-up practices, microfluidic devices have been introduced to improve biomarkers isolation efficiency and specificity. Nonetheless, implementation of lab-on-a-chip platforms in clinical practice may face some challenges, considering its recent application to liquid biopsies. In this review, recent advances and strategies for the use of liquid biopsies in LCa management are discussed, focusing on high-throughput microfluidic devices applied for CTCs and ctDNA isolation and detection, current clinical validation studies and potential clinical utility.

Langerhans Cells Express IL-17A in the Epidermis of Chromoblastomycosis Lesions.

Chromoblastomycosis (CBM) is a chronic fungal infection that affects skin and subcutaneous tissue, and little is known about the immunological aspects of such lesions. We have previously described the high expression of IL-17 in this group. Understanding the innate immune response of patients with CBM would improve the knowledge of its immunopathogenesis and contribute to the most appropriate therapies. Nineteen biopsies of verrucous form were obtained from patients with clinical and histopathological diagnosis of CBM, without treatment. This was done with a double immunostaining with conventional immunohistochemistry and immunofluorescence technique as well as confocal microscopy to detect Langerin and IL-17 expression. All of the specimens that were analyzed showed expression of Langerin in the epidermis - the same as the control group. However, only the CBM group presented cells expressing CD207 in the dermis. Interestingly, the coexpression of IL-17 and Langerin was visualized along the epidermis and dermis in 100% of the lesion group. We demonstrated for the first time in situ coexpression of IL-17 and Langerin (CD207) in epidermal cells of patients with CBM and speculated on their role as IL-17-producing cells or whether they could be a new subpopulation of dendritic cells distinct from Langerhans cells.

Constitutive expression of genes encoding notch receptors and ligands in developing lymphocytes, nTreg cells and dendritic cells in the human thymus.

The thymus is the site of T cell maturation. Notch receptors (Notch1-4) and ligands (DLL1-3 and Jagged1-2) constitute one of several pathways involved in this process. Our data revealed differential constitutive expression of Notch genes and ligands in T lymphocytes and thymic dendritic cells (tDCs), suggesting their participation in human thymocyte maturation. nTreg analyses indicated that the Notch components function in parallel to promote maturation in the thymus.

Transforming growth factor ß and apoptosis in leprosy skin lesions: possible relationship with the control of the tissue immune response in the Mycobacterium leprae infection.

The course of leprosy depends of the host immune response which ranges from the lepromatous pole (LL) to the tuberculoid pole (TT). A comparative study was conducted in 60 patients with the LL and TT. The results showed a mean expression of TGF-ß of 339 ± 99.4 cells/field for TT and of 519.2 ± 68.2 cells/field for LL. Frequency of apoptosis was 6.3 ± 1.8 in TT and 14.0 ± 6.1 in LL. A correlation (p = 0.0251) between TGF-ß and caspase-3 in the LL was found. This finding indicates a role of TGF-ß and apoptosis in the immune response in leprosy.

Paracoccidioidomycosis: cells expressing IL17 and Foxp3 in cutaneous and mucosal lesions.

We demonstrated and quantified by immunohistochemistry the population of cells expressing IL17 and Foxp3 in cutaneous and mucosal paracoccidioidomycosis lesions, associating these populations of cells with different presentations of granulomatous response. For this purpose, 61 skin biopsies and 55 oral mucosal biopsies were evaluated. Cells expressing IL17 were distributed in the inflammatory infiltrate in both groups of lesions and were found in the vessels' wall too. Foxp3+ expression was limited to the nuclei of lymphocytes in the inflammatory infiltrate. The distribution of IL17 was similar among the groups; however, Foxp3+ cells were increased in mucosal lesions that displayed compact granulomas. The results suggest that IL17 seems to play a role in paracoccidioidomycosis cutaneous and mucosal lesions, probably as secondary cells in the clearance of the fungal antigens. The presence of Foxp3+ cells both in skin and mucosa corroborates some previous researches that suggest the role of this group of cells in the modulation of local immune response.

CD1a and factor XIIIa immunohistochemistry in leprosy: a possible role of dendritic cells in the pathogenesis of Mycobacterium leprae infection.

Leprosy is a curable chronic granulomatous infectious disease caused by the bacillus Mycobacterium leprae. This organism has a high affinity for skin and peripheral nerve cells. In the evolution of infections, the immune status of patients determines the disease expression. Dendritic cells are antigen-presenting cells that phagocytose particles and microorganisms. In skin, dendritic cells are represented by epidermal Langerhans cells and dermal dendrocytes, which can be identified by expression of CD1a and factor XIIIa (FXIIIa). In the present study, 29 skin samples from patients with tuberculoid (13 biopsies) and lepromatous (16 biopsies) leprosy were analyzed by immunohistochemistry using antibodies to CD1a and FXIIIa. Quantitative analysis of labeling pattern showed a clear predominance of dendritic cells in tuberculoid leprosy. Difference between the number of positive cells of immunohistochemistry for the CD1a and FXIIIa staining observed in this study indicates a role for dendritic cells in the cutaneous response to leprosy. Dendritic cells may be a determinant of the course and clinical expression of the disease.

In situ immune responses to interstitial pneumonitis in human visceral leishmaniasis.

Lung disease during active human visceral leishmaniasis is frequently reported. As such, studies have associated pulmonary symptoms to interstitial pneumonitis with a mononuclear infiltrate. However, the immune response in this condition has never been described before. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of lung involvement (IPL) in human visceral leishmaniasis. Quantitative methods of analysis were performed using immunohistochemistry, and were compared with a control group of normal lung. Interstitial macrophages and cd8 cells were increased in IPL, and IL-4 as well as TNF-alpha displayed increased expression when compared to the control group. This inflammatory process with a Th2 pattern, as suggested by increased IL-4 and low IFN-gamma expression, is consistent with the immune response in other organs of visceral leishmaniasis. The microenvironment of the immune response in this condition is associated with lung disease in patients with interstitial pneumonitis related to visceral leishmaniasis, increasing the chance of bacterial infection.

Human visceral leishmaniasis expresses Th1 pattern in situ liver lesions.

The architectural and infiltrate pattern of liver human visceral leishmaniasis (HVL) have been systematically classified as typical, fibrogenic or nodular. Despite this histopathological classification, the immune response based on cytokines and cellular phenotypes have never been performed. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of the nodular involvement of the liver in HVL. We evaluated nine cases of the nodular form of HVL. In situ immune response was studied through cytokine analysis and immunohistochemical study for phenotype markers: IL-1, IL-4, IL-10, TNF-alpha, IFN-gamma, CD4+ T cells, CD8+ T cells, CD20, CD68, CD57 and macrophage activation was determined by evaluation of iNOS activity. HVL seems to be related to a better immune response. Amastigotes were rarely found on liver sections. Leishmania antigen expression was also rare and located in the inflammatory nodules. The lower expression of IL-4 and IL-10, moderate expression of TNF-alpha and IFN-gamma demonstrate a panorama of Th1 phenotype. The increased expression of NK cells could help in sustaining this model of response. This pattern of immune response is probably responsible for improvement in the parasite's clearance from liver tissue and it is a prognostic marker of human visceral leishmaniasis.

Hepatic ultrastructural mitochondrial changes prior to antiretroviral therapy in HIV-infected patients in Brazil.

The use of antiretroviral (ARV) medications has been linked to the emergence of severe adverse effects, including mitochondrial toxicity. The liver also appears to be among the affected organs. Nevertheless, different studies suggest that these patients' mitochondrial alterations could be associated to other etiological factors. The goal of this study was to analyze hepatic mitochondria ultrastructural changes in HIV-infected patients under investigation for hepatopathy. Semiquantitative analysis of mitochondria was performed in liver biopsies from 10 patients divided into 2 groups: Group 1 consisted of 5 patients who had never used ARV medications; group 2 consisted of 5 patients who reported previous use of either zidovudine or didanosine. Significant mitochondrial alterations were found in both groups. The summation of the mitochondrial alterations was higher in group 1 (P < .05) when compared with those who had previously used ARV medications. Therefore, the authors conclude that severe mitochondrial alterations occur in HIV-infected patients who have never been submitted to antiretroviral therapy.

Isolated lymphadenitis due to Histoplasma capsulatum diagnosed by fine-needle aspiration biopsy and immunohistochemistry.

In the disseminated form of histoplasmosis, isolation and further identification of Histoplasma capsulatum can be performed by several methods, namely, bone marrow aspiration, blood culture, and liver biopsy. Lymph node disease usually is diagnosed by excisional biopsy. Although fungal stains can identify this fungus, detection of specific antigens by immunohistochemistry shows a higher specificity and sensitivity. This approach can use the cell block method when the material is not sent to fungal cultures or fresh staining.

Isolated lymphadenitis due to Histoplasma capsulatum diagnosed by fine-needle aspiration biopsy and immunohistochemistry

El aislamiento y la posterior identificación de Histoplasma capsulatum en lahistoplasmosis diseminada puede llevarse a cabo por diversos métodos,como la aspiración de médula ósea, el hemocultivo o la biopsia de hígado.La linfadenopatía es habitualmente diagnosticada por extirpación del ganglioafectado. Aunque la tinción del hongo puede llevar a su identificación,la detección de antígenos específicos mediante procedimientos deinmunohistoquímica muestra una mayor sensibilidad y especificidad.Este método permite la fijación de las células cuando el material no va a serprocesado para cultivo micológico o tinción en fresco(AU)

In the disseminated form of histoplasmosis, isolation and further identificationof Histoplasma capsulatum can be performed by several methods, namely,bone marrow aspiration, blood culture, and liver biopsy. Lymph node diseaseusually is diagnosed by excisional biopsy. Although fungal stains can identifythis fungus, detection of specific antigens by immunohistochemistry shows ahigher specificity and sensitivity. This approach can use the cell block methodwhen the material is not sent to fungal cultures or fresh staining(AU)

Immunological parameters of visceral leishmaniasis of Leishmania major-infected golden hamsters.

We can consider the Leishmania major-infected hamster as an interesting model of visceral leishmaniasis. Every hamster infected with L. major strain 70 by peritoneal route developed visceral dissemination of the parasite. When immunological parameters were considered, we saw data quite similar to those presented by visceral leishmaniasis patients: negative leishmanin skin test and presence of anti-leishmania antibody. Histopathological analysis showed dissemination of the parasite mainly to liver and spleen. The former organ showed hypertrophy and hyperplasia of Kupffer cells with focal areas of inflammatory infiltration in nodular pattern. The spleen disclosed intense proliferation and enlargement of mononuclear phagocytic cells, sometimes revealing nodular configuration. Anti-leishmania antibodies were easily detected by indirect immunofluorescent technique in this model. Immunomodulation by Cyclophosphamide decreased the anti-leishmania antibody and delayed-type hypersensitivity test results suggested that hamster was able to develop reaction to leishmania antigen, although leishmanin skin test was negative in the L. major-infected animals. We consider the L. major-infected hamster a useful model for visceral leishmaniasis study because of the similarity of immunological reactions to parasite antigen in human disease.