Juarez Antonio Simões Quaresma

In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus

Zika virus (ZIKV) has recently caused a pandemic disease, and many cases of ZIKV infection in pregnant women resulted in abortion, stillbirth, deaths and congenital defects including microcephaly, which now has been proposed as ZIKV congenital syndrome. This study aimed to investigate the in situ immune response profile and mechanisms of neuronal cell damage in fatal Zika microcephaly cases. Brain tissue samples were collected from 15 cases, including 10 microcephalic ZIKV-positive neonates with fatal outcome and five neonatal control flavivirus-negative neonates that died due to other causes, but with preserved central nervous system (CNS) architecture. In microcephaly cases, the histopathological features of the tissue samples were characterized in three CNS areas (meninges, perivascular space, and parenchyma). The changes found were mainly calcification, necrosis, neuronophagy, gliosis, microglial nodules, and inflammatory infiltration of mononuclear cells. The in situ immune response against ZIKV in the CNS of newborns is complex. Despite the predominant expression of Th2 cytokines, other cytokines such as Th1, Th17, Treg, Th9, and Th22 are involved to a lesser extent, but are still likely to participate in the immunopathogenic mechanisms of neural disease in fatal cases of microcephaly caused by ZIKV.

Zika virus epidemic in Brazil. I. Fatal disease in adults: Clinical and laboratorial aspects

BACKGROUND Zika virus (ZIKV) was first detected in Brazil in May 2015 and the country experienced an explosive epidemic. However, recent studies indicate that the introduction of ZIKV occurred in late 2013. Cases of microcephaly and deaths associated with ZIKV infection were identified in Brazil in November, 2015. OBJECTIVES To determine the etiology of three fatal adult cases. STUDY DESIGN Here we report three fatal adult cases of ZIKV disease. ZIKV infection in these patients was confirmed by cells culture and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and by antigen detection using immunohistochemical assay. Samples of brain and other selected organs taken at autopsy from three patients were also analyzed by histopathological and immunohistological examination. RESULTS The first patient, a 36-year-old man with lupus and receiving prednisone therapy, developed a fulminant ZIKV infection. At autopsy, RT-qPCR of blood and tissues was positive for ZIKV RNA, and the virus was cultured from an organ homogenate. The second patient, a previously healthy female, 16 years of age, presented classic symptoms of Zika fever, but later developed severe thrombocytopenia, anemia and hemorrhagic manifestations and died. A blood sample taken on the seventh day of her illness was positive RT-PCR for ZIKV RNA and research in the serum was positive for antinuclear factor fine speckled (1/640), suggesting Evans syndrome (hemolytic anemia an autoimmune disorder with immune thrombocytopenic purpura) secondary to ZIKV infection. The third patient was a 20-year-old woman hospitalized with fever, pneumonia and hemorrhages, who died on 13days after admission. Histopathological changes were observed in all viscera examined. ZIKV antigens were detected by immunohistochemistry in viscera specimens of patients 1 and 3. These three cases demonstrate other potential complications of ZIKV infection, in addition to microcephaly and Guillain-Barre syndrome (GBS), and they suggest that individuals with immune suppression and/or autoimmune disorders may be at higher risk of developing severe disease, if infected with ZIKV.

Immunity and immune response, pathology and pathologic changes: progress and challenges in the immunopathology of yellow fever.

Yellow fever is a viral hemorrhagic fever, which affects people living in Africa and South America and is caused by the yellow fever virus, the prototype species in the Flavivirus genus (Flaviviridae family). Yellow fever virus infection can produce a wide spectrum of symptoms, ranging from asymptomatic infection or oligosymptomatic illness to severe disease with a high fatality rate. In this review, we focus in the mechanisms associated with the physiopathology of yellow fever in humans and animal models. It has been demonstrated that several factors play a role in the pathological outcome of the severe form of the disease including direct viral cytopathic effect, necrosis and apoptosis of hepatocyte cells in the midzone, and a minimal inflammatory response as well as low‐flow hypoxia and cytokine overproduction. New information has filled several gaps in the understanding of yellow fever pathogenesis and helped comprehend the course of illness. Finally, we discuss prospects for an immune therapy in the light of new immunologic, viral, and pathologic tools. Copyright

Immunopathogenesis of dengue hemorrhagic fever: Contribution to the study of human liver lesions

Dengue infection is an important tropical disease worldwide. The host immune response has been studied in order to better understand lesion mechanisms. It was performed an immunohistochemical study in 14 specimens of liver from patients with dengue hemorrhagic fever (DHF) to characterize cytokines and some factors present in liver lesions and their possible role in the pathogenesis of hepatic injury. Portal tract and hepatic acinus presented high expression of TLR2, TLR3, IL6, and granzyme B. Hepatic acinus also presented iNOS, IL18, and TGF‐beta. Cells expressing IL12, IL13, JAk1, STAT1, and NF‐κB were rarely visualized. Treg cells foxp3+ were absent. TLR2 and TLR3 seem to participate in cellular activation and cytokine production. Cytotoxic response seems to play a role. Although TGF‐beta promotes the activation of Foxp3+ regulatory T cells, IL6 can significantly suppresses their generation. The expression of Treg cells is diminished probably as a result of the high frequency of these cytokines. Both cytokines play a role in the increased vascular permeability and edema observed in dengue liver specimens, with consequent plasma leakage and severity of the disease. It was observed a regular expression of IL‐18 in hepatocytes and lymphocytes of the inflammatory infiltrate in portal tract, which reflects the acute inflammatory response that occurs in the liver and contributes to hepatic injury. At least in part, the increased number of cells expressing IL‐18 could play a role of “up” regulation of FasL and correlate to the phenomenon of apoptosis, a mechanism of destruction of hepatocytes in DHF. J. Med. Virol. 86:1193–1197, 2014.

Immunopathogenesis of HTLV-1-assoaciated myelopathy/tropical spastic paraparesis (HAM/TSP)

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Only a limited percentage of infected individuals develop disease in response to the virus while the majority remain asymptomatic, and HAM/TSP is the most common clinical manifestation of the virus. HAM/TSP is an inflammatory disease of the central nervous system (CNS); however, the mechanism by which HTLV-1 induces HAM/TSP is not yet clear. CD4(+) T lymphocytes are the main reservoirs of HTLV-1 in vivo and perform an important role in the immunological response to this retrovirus. This virus-host interaction may provoke changes in the immunological response, such as the enhanced production of inflammatory cytokines and the spontaneous proliferation of T CD4(+) lymphocytes, which are implicated in the pathogenesis of HAM/TSP.

Prevalência de infecção genital pelo HPV em populações urbana e rural da Amazônia Oriental Brasileira

Foram investigados a prevalencia e os fatores associados a infeccao genital pelo HPV em mulheres de populacao urbana e rural de duas regioes da Amazonia Oriental brasileira. Foi um estudo transversal com 444 mulheres submetidas ao rastreamento para câncer cervical, sendo 233 urbanas e 211 rurais, de janeiro de 2008 a marco de 2010. Coletaram-se amostras da cervice uterina para a pesquisa de DNA do HPV pela PCR. Todas responderam a um formulario epidemiologico. Analise bivariada e por regressao logistica foram empregadas na investigacao dos fatores associados ao HPV. A prevalencia geral de HPV foi de 14,6%. Entre as populacoes, nao houve diferenca significativa, 15% urbana e 14,2% rural. O unico fator de risco explorado no estudo significativamente associado ao HPV foi a situacao conjugal de mulheres residentes na zona rural na faixa de 13 a 25 anos, com maior prevalencia de infeccao entre solteiras, separadas ou viuvas. Conclui-se que, apesar das prevalencias entre as populacoes serem semelhantes, as estrategias preventivas a serem aplicadas seriam especificas para cada populacao.

HIV and lacaziosis, Brazil.

To the Editor: Jorge Lobo disease (lacaziosis) is a chronic deep mycosis for which prognosis is good in terms of survival but unclear in terms of regression of the lesions (1). No involvement of internal organs or mucous membranes is observed. The causative agent is Lacazia loboi (2), a fungus of uncertain phylogeny, which causes an inflammatory infiltrate accompanied by the formation of a granuloma in which giant cells phagocytose a larger number of fungi (3,4). Pecher and Funchs suggested that patients with lacaziosis have a cellular immunodeficiency (5). The disease is more frequent in men and persons 21–40 years of age. It is found exclusively in Latin America; only 1 case has been diagnosed in Europe, and that was due to accidental contamination with material from a dolphin (4). Trauma and injuries or sites of insect bites facilitate penetration of the fungus. Lesion progression is slow, with new lesions arising by contiguity with other lesions or through the lymphatic route (6,7). Clinically, lacaziosis manifests as keloidal lesions of solid consistency and variable size that contain small scales and crusts (6). The lesions are most frequently located in the auricle and on the upper and lower limbs. Cutaneous dissemination of the disease is observed in a relatively small number of cases. We describe a patient with Jorge Lobo disease. The patient was a 59-year-old man, a storeroom employee, who was seen at the Tropical Medical Center in Belem, Brazil, in April 2004. A papula had developed near his right knee in 1992 after a wood splinter had penetrated the skin. The lesion increased in size, and a histopathologic diagnosis of Jorge Lobo disease was made. The lesion was then surgically removed. Approximately 2 years later, the lesion recurred. The patient then went to a dermatology service and was treated with clofazimine, after which the lesion disappeared. However, the lesion reappeared 1 year later. HIV serologic analysis was performed in 2002, and the results were positive. The patient then began treatment for HIV infection. He is currently being monitored at the specialized referral unit in Belem. He does not have any opportunistic infections and is not taking any antiretroviral drugs. The patient came to the dermatology service of the Tropical Medical Center, where dermatologic and histopathologic examinations were conducted and CD cell counts and HIV viral load were measured. Dermatologic aspects of the lesion included an erythematous-infiltrated, hypertrophic plaque with a verrucous surface ≈4 cm long in the distal third of the medical aspect of the right thigh (Figure). A punch biopsy specimen of brown smooth skin 0.35 cm in diameter in an epidermal disk was fixed in formalin. Microscopy of skin sections containing epidermis showed compact keratinization, parakeratotic foci, and irregular hyperplasia with a pseudoepitheliomatous area. A highly dense, nodular, diffuse inflammatory infiltrate was observed at all levels of the dermis. It consisted of macrophages and numerous multinucleated cells, most of them of the foreign body type. Fibroplasia was also noted. Abundant, round parasitic elements surrounded by a double membrane and containing a basophilic nucleus were found in tissues, as well as other anucleated, intracellular, and free parasites that formed chains of >2 cells (Figure A1). Jorge Lobo disease was diagnosed. Laboratory results showed 146 CD4 cells/μL, 251 CD8 cells/μL, a CD4:CD8 ratio of 0.42, and 60,000 copies of HIV viral RNA/mL. Figure Erythematous-infiltrated, hypertrophic plaque with a verrucous surface ≈4 cm long in the distal third of the medical aspect of the right thigh of the patient. Since a cytotoxic response is observed in Jorge Lobo disease (7), HIV infection may increase the susceptibility to infection with L. loboi. Patients with AIDS show a predisposition to diverse fungal infections that classically affect different organs and systems. An association between Jorge Lobo disease and AIDS has not been reported. However, since Jorge Lobo disease is restricted to specific areas of the world and the number of AIDS cases is increasing, especially in Latin America, a possible correlation between HIV infection and Jorge Lobo disease should be considered because of the associated cellular immunodeficiency. The patient showed no signs of other opportunistic infections classically associated with AIDS, and he was not taking any antiretroviral drugs. His initial infection manifested as cutaneous lesions that occur in Jorge Lobo disease. Despite the cellular immunodeficiency, we did not observe atypical dissemination of the lesions. Further studies should be conducted to evaluate the relationship between the cellular immunosuppression of AIDS and secondary infection with L. loboi. In addition, epidemiologic studies are needed to determine the association of AIDS with Jorge Lobo disease.

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 expression of M2 macrophage subpopulation in leprosy skin lesions

The clinical manifestations of the leprosy depend on host immune response and the macrophages are the primary cells involved in this process. M1 and M2 cells exhibited distinct morphology, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophages express receptors such as CD163, CD68, CD206, and costimulatory molecules such as CD80 and CD86, and cytokines that trigger a suppressive or inflammatory response. Thirty-three untreated patients were selected, 17 patients had the tuberculoid leprosy (TT) and 16 had the lepromatous leprosy (LL). We performed immunohistochemistry to detect IL-13, IL-10, TGF-β, FGF-β, CD163, CD68, arginase 1. M2 macrophages showed significant differences between the groups studied with increase in the expression of costimulatory molecules (CD68 and CD163), arginase 1 and cytokines (IL-10, IL-13, TGF-β and FGF-b) in the LL form. Response of M2 macrophages emerge as an alternative for a better understanding of the innate immunity in the polar forms of leprosy, highlighting the role of cytokines, arginase 1 and costimulatory molecules in the repair and suppressive responses in the lepromatous form of the disease.

Characterization of Minaçu virus (Reoviridae: Orbivirus) and pathological changes in experimentally infected newborn mice.

Minaçu virus was isolated from Ochlerotatus scapularis (Diptera: Culicidae) in Minaçu, Goiás State, Brazil, in 1996. In attempting characterization of virus serological (hemagluttination inhibition, HI; indirect immunofluorescence assay, IFA), physicochemical [test for deoxycholate acid (DCA) sensitivity; polyacrylamide gel electrophoresis (PAGE)] tests and ultrastructural studies were made. Virus was also assayed in suckling mice after intracerebral inoculation of 0.02 ml and in VERO and C6/36 cells with 0.1 ml of viral suspension containing 105 LD50/ml. Inoculated and control systems were observed daily. Every 24 h, one control and two inoculated animals were killed for tissue testing, including histopathological changes by haematoxylin and eosin (HE)‐stained sections, which were semi‐quantified. Research into viral antigen in the tissues of mice [central nervous system (CNS), liver, heart, lungs, spleen and kidneys] was carried out by the immunohistochemical technique using the peroxidase system. The virus only replicated in VERO cells, with antigen positive by IFA. Positive complement fixation tests were only obtained using antiserum of Minaçu virus. Minaçu virus is DCA resistant; haemagglutinating activity was negative. By electronic microscopy non‐enveloped virus particles were 75 nm in diameter. PAGE analysis showed Minaçu virus genome profile with 10 RNA segments. Infected, non‐killed animals died 7 days after inoculation. Tissue lesions were observed in all organs, except the lungs. Intense lesions were observed in the CNS and the heart, where neurone and cardiocyte necroses, respectively, were noted. The liver, spleen and kidneys had moderate tissue changes. Viral antigens were more abundant in the CNS and the heart, and absent in the lungs. In conclusion, Minaçu virus belongs to the family Reoviridae, genus Orbivirus.