Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2′-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Skin Prick Tests and Allergy Diagnosis

Skin testing remains an essential diagnostic tool in modern allergy practice. A signifi cant variability has been reported regarding technical procedures, interpretation of results and documentation. This review has the aim of consolidating methodological recommendations through a critical analysis on past and recent data. This will allow a better understanding on skin prick test (SPT) history; technique; (contra-) indications; interpretation of results; diagnostic pitfalls; adverse reactions; and variability factors.

Human herpesvirus-7 as a cause of exanthematous ilnesses in Belém, Pará, Brazil

We screened sera from 370 patients suffering from exanthematous illnesses in Belém, North Brazil, for the presence of human herpesvirus-7 (HHV-7) IgM and IgG antibodies. Samples were obtained from January 1996 to December 2002 and were processed by a HHV-7-specific indirect immunofluorescence assay (IFA). HHV-7-specific IgM and/or IgG antibodies were found in 190 (51.4%) of these patients, with similar prevalence rates (IgM+ and IgG+ subgroups taken together) for female and male subjects: 52.5% and 50.3%, respectively. Serological status as defined by IgG was identified in 135 (36.5%) patients. In 55 (14.9%) of the patients HHV-7 IgM antibodies were detected. HHV-7 IgM- and- IgG antibody rates were similar (p > 0.05) when male and female subjects are compared: 14.4% versus 15.3% and 38.1% versus 35.0%, respectively. Statistically significant difference (p = 0.003) was noted when HHV-7-IgM-positive female and male patients aged 5-8 months are compared. Prevalence rates ranging from 4.6% (female, 5-8 months of age) to 93.3% (female, > 10 years of age) and 12.2% (male, 5-8 months) to 80.0% (male, 8-10 years of age) were noted in the IgG- positive subgroups. A subgroup (n = 131) of patients with IgM or IgG HHV-7 antibodies were examined for the presence of DNA using a polymerase chain reaction/nested PCR. Recent/active HHV-7 infection occurred at a rate of 11.0% (6/55) among patients whose samples presented IgM+ specific antibodies. In a subgroup (n = 76) of patients with high HHV-7-IgG antibody levels (titre > 1:160) DNA could not be detected in sera examined by PCR/nested PCR. Of the six recent/active infections, four subjects with less than 1 year and two with 3 and 6 years of age, presented typical exanthem subitum (E.S), as defined by higher fever (> 38.0 ºC) with duration of 24 to 72 hours, followed by a maculopapular skin rash. Our results underscore the need for searching HHV-7 infection in patients with exanthematous diseases, particularly those presenting with typical E.S. HHV-7 appears therefore to emerge as a newly recognized pathogen of exanthem in our region.

Lesões Inalatórias no Doente Queimado

Inhalation injuries are currently the factor most responsible for mortality in thermally injured patients. Inhalation injuries may occur independently, but generally occur together with skin burn. Smoke inhalation affects all levels of the respiratory system and the extent of the inhalation injury depends on the duration, exposure, amount and toxicity of the fume temperature, concentration and solubility of toxic gases, the occurrence of the accident in a closed space and pre-existing diseases. Smoke inhalation also induces changes in the systemic organs with the need for more fluid for resuscitation. Systemic vasoconstriction, with an elevation in systemic vascular resistance, a fall in myocardial contractility and a great increase in lymphatic flow in soft tissue are the most important changes in systemic organs. On admission of a burn patient there is a high suspicion of inhalation injury when there are signs and symptoms such as hoarseness, strides, dyspnea, carbonaceous sputum, anxiety or disorientation, with or without face burns. The patient with these findings has partial airway obstruction and there is substantial risk complete airway obstruction occurring of secondary to the edema. Patients with suspected inhalation injury should be intubated so as to maintain airway patency and avoid a total obstruction. This group of patients frequently develop respiratory failure with the need for mechanical ventilatory support. Nosocomial infections, sepsis and multiple organ system failure may occur. Late complications of inhalation injury are tracheitis, tracheal stenosis or tracheomalacia and chronic airway disease, which is relatively rare. Early diagnosis of inhalation injury and treatment in a Burn Unit by a group of highly motivated clinicians and a good team of nurses is essential in order to decrease the morbidity and mortality related to inhalation injury.

Evolution of genotypes of Group A streptococci from colonization and infection

Dissertação para obtenção do Grau de Doutor em Biologia

Electrical Skin Phenomena: A Fractional Calculus Analysis

The internal impedance of a wire is the function of the frequency. In a conductor, where the conductivity is sufficiently high, the displacement current density can be neglected. In this case, the conduction current density is given by the product of the electric field and the conductance. One of the aspects the high-frequency effects is the skin effect (SE). The fundamental problem with SE is it attenuates the higher frequency components of a signal. The SE was first verified by Kelvin in 1887. Since then many researchers developed work on the subject and presently a comprehensive physical model, based on the Maxwell equations, is well established. The Maxwell formalism plays a fundamental role in the electromagnetic theory. These equations lead to the derivation of mathematical descriptions useful in many applications in physics and engineering. Maxwell is generally regarded as the 19th century scientist who had the greatest influence on 20th century physics, making contributions to the fundamental models of nature. The Maxwell equations involve only the integer-order calculus and, therefore, it is natural that the resulting classical models adopted in electrical engineering reflect this perspective. Recently, a closer look of some phenomas present in electrical systems and the motivation towards the development of precise models, seem to point out the requirement for a fractional calculus approach. Bearing these ideas in mind, in this study we address the SE and we re-evaluate the results demonstrating its fractional-order nature.

Clinical and epidemiological aspects of HTLV-II infection in São Paulo, Brazil: presence of Tropical Spastic Paraparesis/HTLV-Associated Myelopathy (TSP/HAM) simile diagnosis in HIV-1-co-infected subjects

In this study, the epidemiological and clinical features observed in solely HTLV-II-infected individuals were compared to those in patients co-infected with HIV-1. A total of 380 subjects attended at the HTLV Out-Patient Clinic in the Institute of Infectious Diseases "Emilio Ribas" (IIER), São Paulo, Brazil, were evaluated every 3-6 months for the last seven years by infectious disease specialists and neurologists. Using a testing algorithm that employs the enzyme immuno assay, Western Blot and polymerase chain reaction, it was found that 201 (53%) were HTLV-I positive and 50 (13%) were infected with HTLV-II. Thirty-seven (74%) of the HTLV-II reactors were co-infected with HIV-1. Of the 13 (26%) solely HTLV-II-infected subjects, urinary tract infection was diagnosed in three (23%), one case of skin vasculitis (8%) and two cases of lumbar pain and erectile dysfunction (15%), but none myelopathy case was observed. Among 37 co-infected with HIV-1, four cases (10%) presented with tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM) simile. Two patients showed paraparesis as the initial symptom, two cases first presented with vesical and erectile disturbances, peripheral neuropathies were observed in other five patients (13%), and seven (19%) patients showed some neurological signal or symptoms, most of them with lumbar pain (five cases). The results obtained suggest that neurological manifestations may be more frequent in HTLV-II/HIV-1-infected subjects than those infected with HTLV-II only.

Candida albicans skin abscess

Subcutaneous candidal abscess is a very rare infection even in immunocompromised patients. Some cases are reported when breakdown in the skin occurs, as bacterial cellulites or abscess, iatrogenic procedures, trauma and parenteral substance abuse. We describe a case of Candida albicans subcutaneous abscess without fungemia, which can be associated with central venous catheter.

Orbital Exenteration for Eyelid Skin Carcinoma

Exenteration of the orbit is a disfiguring and destructive procedure; it is generally performed for orbital malignancies and often provides a significant reconstructive challenge. Our purpose was to evaluate the clinical indications for orbital exenteration in a tertiary referral center and to assess the reconstructive options employed. A retrospective nonrandomized analysis was performed, selecting all patients undergoing orbital exenteration over a 5-year period, between January 2005 and January 2010. Patient demographics, tumor characteristics, and reconstructive techniques used were evaluated. Twenty patients with a mean age of 76.5 years underwent total orbital exenteration. Basal cell carcinoma was the main operative indication (45%), followed by squamous cell carcinoma (15%). Reconstructive techniques included cover of the raw orbital cavity with a temporal muscular flap in all cases followed with split skin grafting (25%), bilaterally pedicle V-Y advancement flap (10%) and a fasciocutaneous island flap of the retroauricular region (65%). Twenty percent of patients had local complications and all were treated in a satisfactory fashion. Eyelid skin tumors remain an important cause of orbital exenteration. Temporal muscle flap is a reliable and stable reconstructive solution after orbital exenteration and additional aid is supplied with skin grafts or local flaps. This technique ensures a good aesthetic outcome and better situation for later complementary treatments and minimal associated donor site morbidity.

Characterization and Biological Activities of Ocellatin Peptides from the Skin Secretion of the Frog Leptodactylus pustulatus

Eight new peptides were isolated from the skin secretion of the frog Leptodactylus pustulatus and their amino acid sequences determined by de novo sequencing and by cDNA cloning. Structural similarities between them and other antimicrobial peptides from the skin secretion of Leptodactylus genus frogs were found. Ocellatins-PT1 to -PT5 (25 amino acid residues) are amidated at the C-terminus, while ocellatins-PT6 to -PT8 (32 amino acid residues) have free carboxylates. Antimicrobial activity, hemolytic tests, and cytotoxicity against a murine fibroblast cell line were investigated. All peptides, except for ocellatin-PT2, have antimicrobial activity against at least one Gram negative strain. Ocellatin-PT8 inhibited the growth of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella choleraesuis strains with MICs in the 60−240 μM range. No significant effect was observed in human erythrocytes and in a murine fibroblast cell line after exposure to the peptides at MICs. A comparison between sequences obtained by both direct HPLC-MS de novo sequencing and cDNA cloning demonstrates the secretion of mature peptides derived from a pre-pro-peptide structure.

Annular Lichen Planus in Association with Crohn Disease

BACKGROUND: Lichen planus is an idiopathic inflammatory disease of the skin and mucous membranes. Although the etiology is not established, it has been associated with autoimmune diseases, viral infections, drugs and dental restoration materials. However, the association with inflammatory bowel disease has been very rarely reported in the literature. CASE REPORT: A 19-year-old female patient presented with annular lesions on her upper body and limbs, with a sharply defined border and non-atrophic skin in the center. The lesions were hyperpigmented and had been stable for over one year. The histopathology confirmed the diagnosis of annular lichen planus. She had weight loss, occasional diarrhea, and a severe anemia. The investigation of these symptoms led to the diagnosis of Crohn disease and a sickle cell trait. Therapy with systemic corticosteroids and mesalazine controlled the intestinal disease, with concomitant improvement of the skin lesions. CONCLUSIONS: As lichen planus can be associated with other immunological disorders, the association with inflammatory bowel disease should be considered in the evaluation of the patient.

American tegumentary leishmaniasis: langerhans cells in montenegro skin test

This work analyzed the histopathology and epidermal Langerhans cells (LC) of Montenegro skin test (MST) in patients with American tegumentary leishmaniasis (ATL) in order to in situ characterize and compare the immunological reaction of the two major clinical forms of ATL, localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). MST histopathology of both LCL and MCL showed superficial and deep perivascular inflammatory infiltrate composed mainly of lymphocytes and histiocytes. Epidermal LC population was higher in MST biopsies taken from LCL patients when compared to MCL group, at 48 and 72 hours after antigen inoculation. Increased number of epidermal LC displayed in MST biopsies of LCL patients represents specific cellular immunity against parasites. The decrease of LC in MST biopsies of MCL patients does not necessarily indicate a worse specific cellular immunity in this clinical form of leishmaniasis.

Cytokine profile in Montenegro skin test of patients with localized cutaneous and mucocutaneous leishmaniasis

American tegumentary leishmaniasis presents as two major clinical forms: localized cutaneous leishmaniasis (LCL) and mucocutaneous leishmaniasis (MCL). The immune response in leishmaniasis is efficiently evaluated by the response to Leishmania antigen through the Montenegro skin test (MST). Both LCL and MCL present positive response to MST, indicating that the patients present cell-mediated immunity against the parasite - Leishmania. In spite of the presence of immunity in MCL, this is not sufficient to stop disease progression and prevent resistance to treatment. In this study we demonstrated interleukin (IL) 2, 4, 5 and interferon (IFN) gamma expression in biopsies of MST of ten patients with American tegumentary leishmaniasis. The obtained results were compared between LCL (n = 5) and MCL (n = 5) patients. The MST of MCL patients displayed a higher expression of IL-2, IL-4 and IL-5, in comparison to LCL. There was no significant difference in IFN-gamma expression between groups. The obtained results suggest the role of IL-4 and IL-5 in the maintenance of the immunopathogenic mechanism of the destructive lesions that characterize MCL.

Drug Provocation Tests to Betalactam Antibiotics: Experience in a Paediatric Setting

Background: Few studies have been performed in children withs uspected betalactam allergy.We aimed to assess the role of the drug provocation test(DPT)with betalactams in a paediatric setting and to study the association between allergy to betalactam antibiotics and other allergic diseases. Methods:We included all the patients under 15 years old who were consecutively referred to the Immunoallergy Department, Dona Estefânia Hospital,Portugal(January 2002 to April 2008)for a compatible history of allergic reaction to betalactam. All were submitted to a DPT.Children were proposed to performs kintests(ST)to betalactam antibiotics followed by DPT. If they decline ST,a DPT with the culprit drug was performed. Results: We studied 161 children,60%were boys,with a median age of 5years old at the time of the DPT.Thirty-three patients(20.5%)had an immediate reaction and 33(20.5%)a non-immediate reaction. These verity of there porte dreactions was low in most cases. Skin tests to betalactams were performed in 47 children and were positive in 8.DPT was positive inonlyone(3.4%)of the patients skin tested and in 11(13.4%)of those not skin tested. These verity of the DPT reaction was low.Asthma and food allergy were associated with a positive DPT in the later group. Conclusions: DPT seems a safe procedure even in the absence of ST in non-severe cases. This could be a practical optionin infants and pre-school children,where ST are painful and difficult to perform.Additional caution should be taken in children with asthma and food allergy.