Ana Maria de Oliveira Ramos

Pathology of congenital Zika syndrome in Brazil: a case series

BACKGROUND Zika virus is an arthropod-borne virus that is a member of the family Flaviviridae transmitted mainly by mosquitoes of the genus Aedes. Although usually asymptomatic, infection can result in a mild and self-limiting illness characterised by fever, rash, arthralgia, and conjunctivitis. An increase in the number of children born with microcephaly was noted in 2015 in regions of Brazil with high transmission of Zika virus. More recently, evidence has been accumulating supporting a link between Zika virus and microcephaly. Here, we describe findings from three fatal cases and two spontaneous abortions associated with Zika virus infection. METHODS In this case series, formalin-fixed paraffin-embedded tissue samples from five cases, including two newborn babies with microcephaly and severe arthrogryposis who died shortly after birth, one 2-month-old baby, and two placentas from spontaneous abortions, from Brazil were submitted to the Infectious Diseases Pathology Branch at the US Centers for Disease Control and Prevention (Atlanta, GA, USA) between December, 2015, and March, 2016. Specimens were assessed by histopathological examination, immunohistochemical assays using a mouse anti-Zika virus antibody, and RT-PCR assays targeting the NS5 and envelope genes. Amplicons of RT-PCR positive cases were sequenced for characterisation of strains. FINDINGS Viral antigens were localised to glial cells and neurons and associated with microcalcifications in all three fatal cases with microcephaly. Antigens were also seen in chorionic villi of one of the first trimester placentas. Tissues from all five cases were positive for Zika virus RNA by RT-PCR, and sequence analyses showed highest identities with Zika virus strains isolated from Brazil during 2015. INTERPRETATION These findings provide strong evidence of a link between Zika virus infection and different congenital central nervous system malformations, including microcephaly as well as arthrogryposis and spontaneous abortions. FUNDING None.

Osteopenia: a bone disorder associated with diabetes mellitus

Although osteopenia has been associated with human diabetes mellitus, the pathogenesis of diabetic osteopenia is unclear. In the present study, we evaluated the effect of diabetes on histomorphometry, bone mineral density (BMD)—measured by dual-energy X-ray absorptiometry (DXA)—and biomarkers of bone metabolism in rats up to 120 days after the onset of experimental diabetes. Female Wistar rats with a regular estrous cycle were randomly divided into two groups: control rats (n = 15) and diabetic rats without insulin treatment (n = 25). Diabetes was induced by injection of alloxan and was confirmed by the determination of blood glucose concentration (>250 mg/dl). The results revealed an approximate threefold increase of femoral trabecular distance in diabetic rats compared to controls. Conversely, trabecular thickness and bone trabecular volume were reduced twofold and 77%, respectively. BMD in both the metadiaphyseal region and total area of the femur was found to be clearly reduced in diabetic animals, with no significant differences between the groups. Serum alkaline phosphatase (ALP) and tartarate-resistant acid phosphatase (TRAP) activities showed significant six- and twofold increases, respectively, in diabetic rats. There were significant decreases in serum calcium and albumin concentrations in diabetic rats, but no difference was observed in serum magnesium, phosphorus, or creatinine concentrations between the groups. Overall, our findings support the conclusion that the diabetic state is associated with alterations in bone turnover, resulting in the development of osteopenia, which is related to the time of evolution of the disorder.

Reversible vanishing bile duct syndrome induced by carbamazepine.

Carbamazepine, a widely used anticonvulsant, can induce hepatotoxicity, usually evolving with an acute hepatitis that ceases after drug withdrawal. Carbamazepine-induced vanishing bile duct syndrome (VBDS) is a rare complication and has seldom been reported in the medical literature. This report presents a case of a 26-year-old male who had onset of epilepsy at 12 months of age and was initially treated with phenobarbital. Carbamazepine (1200 mg/day) was added in June 1996 when he was 22 years old to control the frequency of seizures. Two years later, during a routine investigation, elevation of serum γ-glutamyltransferase (GGT) levels was detected. For this reason, the patient was weaned off carbamazepine, followed 6 months later by complete withdrawal of the drug. The first liver biopsy disclosed total absence of interlobular bile ducts (IBD) in 30 portal tracts. Fourteen months later, a control biopsy showed the presence of IBD in eight of 14 portal tracts. There was also a decrease of GGT levels detected 27 months after withdrawal of carbamazepine. This case illustrates the ductopenic effect of carbamazepine when used for a prolonged time, as reported in three previous publications. However, this is the first case in which there was a remission of the VBDS and bile duct regeneration after withdrawal of the drug.

Tratamento tópico de queimaduras do dorso de ratos com ácido hialurônico

Previous studies have sugested that hyaluronic acid, a naturally occuring glycosaminoglycan in high doses and for a long time on extracelular matrix of healing wounds of fetus, is the responsible for the result of the fetal healing without scar and wound contraction. The present study investigated the topical effect of hyaluronic acid in burns of adult rats, until the complete epitelization of the lesions. Twenty Wistar rats weighting 225±15g were used. A 5 cm2 back burn was done in each rat under anesthesia. In the I group (n=10) the burns were treated with daily topical application of 1ml of 1% hyaluronic acid and in the II group (n=10) the treatment was done with 1ml of saline 0,9%, until the complete healing. The healing time was 29±1,33 days in the I group and 38±2,58 days in the II group . The histologic score was 27,0±2,78 in the I group and 18,1±3,66 in the II group. The differences were significant (p <0,05). The analysis of the aesthetic result revealed larger deformities in the scars of the II group (controls). In conclusion, the topic hyalurinic acid contributed to accelerate the healing rate, it improved the histologic evolution and turned better the aesthetic result in burns of adult rats.

Leishmaniose visceral fatal associada à síndrome de imunodeficiência adquirida: relato de caso com achados necroscópicos e estudo imuno-histoquímico

A case of fatal visceral leishmaniasis associated with immunodeficiency syndrome in a 32 year-old male patient is reported. The protozoonosis was responsible for the patients death. Visceral leishmaniasis showed itself in an atypical form, at necropsy, with an intense parasitation of the mononuclear phagocitic system and damaging organs not usually affected by the disease, such as the adrenals, the kidneys, the lungs and the brain. Parasitised cells were observed within small vessels in several tissues. An immunohistochemical study was done on samples from the spleen, tymphonodes and brain, showing strong reactivity with antibody directed against leishmania.

Schwannoma da goteira olfatória: relato de caso

Intracranial schwannoma not related to cranial nerves are unusual and rarely found in the subfrontal region. We report a case of olfactory groove schwannoma in a 27-year-old male, who presented with anosmia and headache initiated one year ago. At admission, bilateral papilledema was noted with absense of motor deficits or cranial nerves abnormalities. Cranial computed tomography (CT) revealed a bifrontal multicystic isodense enhancing mass lesion causing a frontal ventricular horn compression. Radiological features resembled that of a cystic olfactory groove meningioma. Decompressive bifrontal craniotomy was done. One month later, CT demonstrated a homogeneously contrast-enhancing mass in the olfactory groove region who extended into the left nasal cavity. Magnetic resonance imaging did not add more informations. A second surgical procedure was done through a nasoethmoidal approach with incomplete resection of the lesion. The complete tumor resection was only possible in a third surgery through another bifrontal approach. The hystopathological diagnosis of schwannoma was performed by conventional methods and confirmed by immunohistoquemical staining for S-100 protein. The rarity of this tumor and his clinical, radiological and histological aspects justify this publication.

Cytokine expression in the duodenal mucosa of patients with visceral leishmaniasis

INTRODUCTION Visceral leishmaniasis (VL) is a neglected tropical disease with a complex immune response in different organs. This pattern of organ-specific immune response has never been evaluated in the gastrointestinal tract. The aim of this study was to determine the in situ immune response in duodenal biopsies on patients with VL. METHODS A case-control study was conducted on 13 patients with VL in comparison with nine controls. The immune response was evaluated using immunohistochemistry, for CD4, CD8, CD68, IL-4, IFN-gamma, TNF-alpha and IL-10. Histological findings from the villi, crypts and inflammatory process were analyzed. RESULTS All the cases of VL presented Leishmania antigens. No antigen was detected in the control group. The villus size was greater in the VL patients (p < 0.05). CD68 (macrophages) and CD4 levels were higher in the VL patients (p < 0.05). No differences in the expression of CD8, TNF-alpha, IL-10 or IL-4 were demonstrated. The number of cells expressing IFN-gamma was lower in the VL patients (p < 0.05). CONCLUSIONS Low levels of cytokines were found in the gastrointestinal tract of patients with VL. This pattern was not found in other organs affected by the disease. Immunotolerance of this tissue against Leishmania could explain these findings, as occurs with intestinal bacteria.

Comparison of prophylactic and therapeutic use of short-chain fatty acid enemas in diversion colitis: a study in Wistar rats

OBJECTIVES: To study the effect of short‐chain fatty‐acids on atrophy and inflammation of excluded colonic segments before and after the development of diversion colitis. INTRODUCTION: Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, possibly evolving with mucous and blood discharge. The most favored hypotheses to explain its development is short‐chain fatty‐acid deficiency in the colon lumen. METHODS: Wistar rats were submitted to colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short‐chain fatty‐acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after post‐operative day 40). The mucosal thickness of the excluded colon was measured histologically. The inflammatory reaction of the mucosal lamina propria and the lymphoid tissue response were quantified through established scores. RESULTS: There was a significant thickness recovery of the colonic mucosa in group B2 animals (p  =  0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p  =  0.0126) and in the intestinal lumen (p  =  0.0256). Group A2 showed no mucosal thickness recovery and significant increases in the numbers of lymphocytes (p  =  0.0006) and eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p  =  0.0022). CONCLUSION: Therapeutic use of short‐chain fatty‐acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy.

Hepatic stellate cell activation and hepatic fibrosis in children with type 1 autoimmune hepatitis: an immunohistochemical study of paired liver biopsies before treatment and after clinical remission

Objectives The activation of hepatic stellate cells (HSC) is considered the most important event in hepatic fibrogenesis. The precise mechanism of this process is unknown in autoimmune hepatitis (AIH), and more evidence is needed on the evolution of fibrosis. The aim of this study was to assess these aspects in children with type 1 AIH. Methods We analyzed 16 liver biopsy samples from eight patients, paired before treatment and after clinical remission, performed an immunohistochemical study with anti-&agr; actin smooth muscle antibody and graded fibrosis and inflammation on a scale of 0–4 (Batts and Ludwig scoring system). Results There was no significant reduction in fibrosis scores after 24±18 months (2.5±0.93 vs. 2.0±0.53, P=0.2012). There was an important decrease in inflammation: portal (2.6±0.74 vs. 1.3±0.89, P=0.0277), periportal/periseptal (3.0±0.76 vs. 1.4±1.06, P=0.0277), and lobular (2.8±1.04 vs. 0.9±0.99, P=0.0179). Anti-&agr; actin smooth muscle antibodies were expressed in the HSC of the initial biopsies (3491.93±2051.48 μm2), showing a significant reduction after remission (377.91±439.47 μm2) (P=0.0117). Conclusion HSC activation was demonstrated in the AIH of children. The reduction of this activation after clinical remission, which may precede a decrease in fibrosis, opens important perspectives in the follow-up of AIH.

Zinc supplementation reduces RANKL/OPG ratio and prevents bone architecture alterations in ovariectomized and type 1 diabetic rats

Type 1 diabetes mellitus (T1DM) and estrogen deficiency are associated with several alterations in bone turnover. Zinc (Zn) is required for growth, development, and overall health. Zinc has been used in complementary therapy against bone loss in several diseases. We hypothesized that Zn supplementation represents a potential therapy against severe bone loss induced by the combined effect of estrogen deficiency and T1DM. We evaluated the protective effect of Zn against bone alterations in a chronic model of these disorders. Female Wistar rats were ramdomized into 3 groups (5 rats each): control, OVX/T1DM (ovariectomized rats with streptozotocin-induced T1DM), and OVX/T1DM+Zn (OVX/T1DM plus daily Zn supplementation). Serum biochemical, bone histomorphometric, and molecular analyses were performed. Histomorphometric parameters were similar between the control and OVX/T1DM+Zn groups, suggesting that Zn prevents bone architecture alterations. In contrast, the OVX/T1DM group showed significantly lower trabecular width and bone area as well as greater trabecular separation than the control. The OVX/T1DM and OVX/T1DM+Zn groups had significantly higher serum alkaline phosphatase activity than the control. The supplemented group had higher levels of serum-ionized calcium and phosphorus than the nonsupplemented group. The RANKL/OPG ratio was similar between the control and OVX/T1DM+Zn groups, whereas it was higher in the OVX/T1DM group. In conclusion, Zn supplementation prevents bone alteration in chronic OVX/T1DM rats, as demonstrated by the reduced RANKL/OPG ratio and preservation of bone architecture. The findings may represent a novel therapeutic approach to preventing OVX/T1DM-induced bone alterations.