Abscopal Effect after Radiosurgery for Solitary Brain Metastasis from Non-small Cell Lung Cancer.

The abscopal effect is a phenomenon relating to the treatment of metastatic cancer in which localized irradiation to a tumor concurrently causes shrinkage of tumors distant from the area of treatment. Localized radiotherapy is thought to cause anti-tumor immunologic responses that lead to regression and remission of cancers distant to the initial location of treatment. We present a 47-year-old male with brain metastasis from non-small cell lung cancer (NSCLC) who went into remission following stereotactic radiosurgery treatment to a brain lesion, in the absence of systemic treatment. We discuss the novelty of this case and its importance to future research on the abscopal effect. Though it is difficult to distinguish the abscopal effect from spontaneous remission of non-targeted cancer, this report sheds insight on the potential for improving treatment for the leading cause of cancer death worldwide.

Assessment of public health risk associated with viral contamination in harvested urban stormwater for domestic applications.

Capturing stormwater is becoming a new standard for sustainable urban stormwater management, which can be used to supplement water supply portfolios in water-stressed cities. The key advantage of harvesting stormwater is to use low impact development (LID) systems for treatment to meet water quality requirement for non-potable uses. However, the lack of scientific studies to validate the safety of such practice has limited its adoption. Microbial hazards in stormwater, especially human viruses, represent the primary public health threat. Using adenovirus and norovirus as target pathogens, we investigated the viral health risk associated with a generic scenario of urban stormwater harvesting practice and its application for three non-potable uses: 1) toilet flushing, 2) showering, and 3) food-crop irrigation. The Quantitative Microbial Risk Assessment (QMRA) results showed that food-crop irrigation has the highest annual viral infection risk (median range: 6.8×10(-4)-9.7×10(-1) per-person-per-year or pppy), followed by showering (3.6×10(-7)-4.3×10(-2)pppy), and toilet flushing (1.1×10(-7)-1.3×10(-4)pppy). Disease burden of each stormwater use was ranked in the same order as its viral infection risk: food-crop irrigation>showering>toilet flushing. The median and 95th percentile risk values of toilet-flushing using treated stormwater are below U.S. EPA annual risk benchmark of ≤10(-4)pppy, whereas the disease burdens of both toilet-flushing and showering are within the WHO recommended disease burdens of ≤10(-6)DALYspppy. However, the acceptability of showering risk interpreted based on the U.S. EPA and WHO benchmarks is in disagreement. These results confirm the safety of stormwater application in toilet flushing, but call for further research to fill the data gaps in risk modeling as well as risk benchmarks.

Fixed sized samples for type-specific surveillance of human papillomavirus in genital warts.

Surveillance data suggest that human papillomavirus (HPV) vaccination in Australia is reducing the incidence of genital warts. However, existing surveillance measures do not assess the proportion of the remaining cases of warts that are caused by HPV types other than 6 or 11, against which the vaccine has no demonstrated effectiveness. Using computer simulation rather than sample size formulae, we established that genotyping at least 60 warts can accurately test whether the proportion of warts due to HPV types not targeted by the vaccine has increased (Type I error probability ≤ 0.05, Type II error probability <0.07). Standard formulae for calculating sample size, in contrast, suggest that a sample size of more than 130 would be required for this task, but using these formulae entails making several strong assumptions. Our methods require fewer assumptions and demonstrate that a smaller sample size than anticipated could be used to address the question of what proportion of post-vaccination cases of warts are due to nonvaccine types. In conjunction with indications of incidence and prevalence provided by existing surveillance measures, this could indicate the number of cases of post-vaccination warts due to nonvaccine types and hence whether type replacement is occurring.

Tracking type specific prevalence of human papillomavirus in cervical pre-cancer: a novel sampling strategy.

BACKGROUND: Surveillance designed to detect changes in the type-specific distribution of HPV in cervical intraepithelial neoplasia grade 3 (CIN-3) is necessary to evaluate the effectiveness of the Australian vaccination programme on cancer causing HPV types. This paper develops a protocol that eliminates the need to calculate required sample size; sample size is difficult to calculate in advance because HPV's true type-specific prevalence is imperfectly known. METHOD: A truncated sequential sampling plan that collects a variable sample size was designed to detect changes in the type-specific distribution of HPV in CIN-3. Computer simulation to evaluate the accuracy of the plan at classifying the prevalence of an HPV type as low (< 5%), moderate (5-15%), or high (> 15%) and the average sample size collected was conducted and used to assess its appropriateness as a surveillance tool. RESULTS: The plan classified the proportion of CIN-3 lesions positive for an HPV type very accurately, with >90% of simulations correctly classifying a simulated data-set with known prevalence. Misclassifying an HPV type of high prevalence as being of low prevalence, arguably the most serious kind of potential error, occurred < 0.05 times per 100 simulations. A much lower sample size (21-22 versus 40-48) was required to classify samples of high rather than low or moderate prevalence. CONCLUSIONS: Truncated sequential sampling enables the proportion of CIN-3 due to an HPV type to be accurately classified using small sample sizes. Truncated sequential sampling should be used for type-specific HPV surveillance in the vaccination era.

Detection of antibodies against Paracoccidioides brasiliensis melanin in in vitro and in vivo studies during infection.

Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.

Involvement of extracellular matrix proteins in the course of experimental paracoccidioidomycosis.

We aimed at determining involvement of extracellular matrix proteins (ECMp) and an ECM-binding adhesin (32-kDa protein) from Paracoccidioides brasiliensis, in the course of experimental paracoccidioidomycosis. BALB/c mice were infected with P. brasiliensis conidia previously incubated with soluble laminin, fibronectin and fibrinogen or a mAb against the fungal adhesin. Inflammatory response, chitin levels and cytokine production at different postinfection periods were determined. Chitin was significantly decreased in lungs of mice infected with ECMp-treated conidia when compared with controls at week 8, especially with laminin and fibrinogen. Contrariwise, when animals were infected with mAb-treated conidia no differences in chitin content were found. The observed inflammatory reaction in lungs was equivalent in all cases. IFN-gamma increased significantly in lungs from mice infected with soluble ECMp - (at day 4 and week 12) or mAb-treated conidia (at week 12) when compared with animals infected with untreated conidia. Significant increased levels of tumour necrosis factor-alpha were observed at 8 weeks in animals infected with ECMp-treated conidia while no differences were observed during the remaining periods. These findings point toward an inhibitory effect of ECMp on P. brasiliensis conidia infectivity and suggest that these proteins may interfere with conidia initial adhesion to host tissues probably modulating the immune response in paracoccidioidomycosis.

Risk stratification of chest pain patients in the emergency department by a nurse utilizing a point of care protocol.

OBJECTIVE: Risk stratification of patients with ischaemic type chest pain assessed in the emergency department utilizing a point of care (POC) protocol. METHODS: Patient demographics, cardiac biomarkers, management and follow-up at 6 months were reviewed for patients seen over 20 months. RESULTS: Out of 546 patients, 351 (64%) were admitted. The diagnoses after admission were confirmed as acute myocardial infarction in 59 patients and unstable angina, (cTroponin T<0.09 ng/ml) in 92 patients. The c-statistic of the receiver operating curves for myocardial infarction (myocardial infarction, cTroponinT at 12 h >0.09 ng/ml) as determined by the POC assay was cTroponin I=0.884, CK-MB=0.883, myoglobin=0.845 and beta-type natriuretic peptide (BNP)=0.755. The c-statistic for the same sample assessed by the hospital laboratory was cTroponin T=0.893: for CK-MB within 12 h of admission it was 0.918; the 12 h cTroponin T was 0.982 and within 24 h of admission NT pro-BNP was 0.789. POC BNP in patients admitted was 68 ng/l (median) vs. 24 ng/l (median) for those not admitted, (P<0.001). POC BNP for patients admitted with unstable angina (12 h cTroponin T <0.09 ng/ml) was 47 ng/l (median, P<0.001). At 6 months, 14 patients had died; five during admission, two within 30 days and seven up to 6 months. During admission two died from heart failure, two with respiratory tract infection and one from carcinoma. Of those not admitted one had died from asbestosis. CONCLUSION: Risk stratification by a specialist nurse utilizing a POC protocol is an appropriate means of assessing patients with chest pain.

Paracoccidioides brasiliensis conidia recognize fibronectin and fibrinogen which subsequently participate in adherence to human type II alveolar cells: involvement of a specific adhesin.

We examined the ability of Paracoccidioides brasiliensis conidia to interact with fibronectin, fibrinogen and with A549 cells, in order to establish the nature of the molecules involved. Conidia bound to immobilized proteins in a concentration-dependent manner. Antibodies against fibronectin and fibrinogen inhibited the fungal adherence to the corresponding proteins; as did laminin and fibronectin, but not fibrinogen when added in soluble form; however, the fibrinogen fragment D interfered with adhesion in a significant manner. Various monosaccharides and RGD/RGDS peptides had no effect on adherence to fibronectin or fibrinogen, while N-acetylneuraminic acid (NANA) abolished adherence to both proteins. Additionally, these proteins were detected on the surface of A549 cells. Inhibition assays showed a significant decrease in fungal adherence when A549 cells were treated with anti-fibrinogen, anti-fibronectin antibodies and a purified adhesin of P. brasiliensis (32-kDa protein); or when conidia were treated with these soluble proteins, mAb anti-32-kDa protein, RGD peptides and NANA. These results suggest that fibrinogen and fibronectin facilitate the adherence of conidia to A549 cells probably through the interaction with adhesin-type molecules or a sialic acid based recognition system. These interactions appear to play a role in the initial fungal attachment to the lung, and consequently, also in the pathogenesis of paracoccidioidomycosis.

Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins.

Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, beta-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.

Combined use of Paracoccidioides brasiliensis recombinant 27-kilodalton and purified 87-kilodalton antigens in an enzyme-linked immunosorbent assay for serodiagnosis of paracoccidioidomycosis.

The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzyme-linked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM. A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied. Eighteen of these patients were also evaluated at every follow-up appointment. Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied. Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera. These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM.

Histoplasma capsulatum synthesizes melanin-like pigments in vitro and during mammalian infection.

Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogen Histoplasma capsulatum var. capsulatum produced melanin or melanin-like compounds in vitro and during infection. Growth of H. capsulatum mycelia in chemically defined minimal medium produced pigmented conidia. Growth of H. capsulatum yeast in chemically defined minimal medium with L-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected with H. capsulatum and from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance to H. capsulatum yeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that H. capsulatum conidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.

Paracoccidioides brasiliensis 87-kilodalton antigen, a heat shock protein useful in diagnosis: characterization, purification, and detection in biopsy material via immunohistochemistry.

The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.