Erratum to: A novel ELISA for diagnosis of Glanzmann's thrombasthenia and the heterozygote carriers.

Erratum to: Annals of Hematology 91(6): 917­921. DOI 10.1007/s00277-011-1390-1 . The authors inadvertently omitted 2 fellow authors from the author list: Dr. Diego Butera should be listed as the fourth author. His affiliation is Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, NSW, Australia. His contributions are as follows: Designed, synthesized and produced EcAPv. He has no competing interests to declare. Dr. Geraldo S. Magalhaes should be listed as the fifth author. His affiliation is Laboratory of Immunopathology, Butantan Institute, São Paulo, SP, Brazil. His contributions are as follows: Produced more EcAPv when requested in October 2009. He has no competing interests to declare.

Unraveling the processing and activation of snake venom metalloproteinases.

Snake venom metalloproteinases (SVMPs) are zinc-dependent enzymes responsible for most symptoms of human envenoming. Like matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAM) proteins, SVMPs are synthesized as zymogens, and enzyme activation is regulated by hydrolysis of their prodomain, but the processing of SVMPs is still unclear. In this study, we attempted to identify the presence of prodomain in different compartments of snake venom glands as zymogens or in the free form to elucidate some mechanism involved in SVMP activation. Using antibodies obtained by immunization with a recombinant prodomain, bands of zymogen molecular mass and prodomain peptides were detected mostly in gland extracts all along the venom production cycle and in the venom collected from the lumen at the peak of venom production. Prodomain was detected in secretory cells mostly in the secretory vesicles near the Golgi. We hypothesize that the processing of SVMPs starts within secretory vesicles and continues in the lumen of the venom gland just after enzyme secretion and involves different steps compared to ADAMs and MMPs but can be used as a model for studying the relevance of peptides resulting from prodomain processing and degradation for controlling the activity of metalloproteinases.

A novel ELISA for diagnosis of Glanzmann's thrombasthenia and the heterozygote carriers.

A sensitive and specific sandwich ELISA was developed for the diagnosis of Glanzmann's thrombasthenia (GT) and the heterozygote carriers of the disease using whole blood platelets. The assay used anti-CD36 antibody to capture platelets from platelet-rich plasma which was subsequently treated with a bioengineered disintegrin/alkaline phosphatase hybrid protein specific for GP IIb/IIIa. The test allows large number of samples to be typed and can also be used on stored samples. The assay correctly diagnosed 40 normal healthy individuals, 10 GT cases, 10 heterozygotes, 3 Bernard-Soulier syndrome cases and 2 type 3 GT cases. ELISA plates were stable at room temperature up to 3 weeks without any loss of activity. This novel and simple test can be widely used for heterozygote detection besides diagnosing GT cases without using a sophisticated flow cytometer or a platelet aggregometer and has wide applicability in countries like India where many of these cases remain undiagnosed due to the lack of diagnostic facilities.

Prevalence of dermatomycosis in a Brazilian tertiary care hospital.

A total of 233 specimens obtained from suspected cases of dermatomycosis from 189 patients were examined for causative fungi from December 2009 to May 2010 in a tertiary care hospital in the city of Belo Horizonte, state of Minas Gerais, southeastern Brazil. Yeast and fungal isolates obtained from specimens were regarded as conclusive diagnosis of mycoses in 82 cases (35.19 %), with the exception of two patients with pityriasis versicolor (2.4 %), in which the diagnosis was made only by direct examination plus clinical diagnostics of individuals. Forty-four subjects (23.28 %) were infected in more than one anatomical site. There was a higher occurrence on female patients (146, 77.2 %) than male (43, 22.8 %). Most of the infected patients were aged between 41 and 70 years (68.29 %). There were no statistically significant differences between occurrence of fungal infection and gender, presence of secondary disease and contact with animals. The largest number of examined material occurred in samples from toenails, which resulted in 50 % of positive cultures. Candida species were the most frequent group causing dermatomycosis in many anatomical sites, mainly in toenails and fingernails. Candida parapsilosis was the most representative (40.24 %) among all agents causing dermatomycosis of toenails and fingernails, followed by Candida tropicalis (20.73 %) and Trichophyton rubrum (10.98 %). Among the dermatophytes, Trichophyton genus represented over 80 % of the isolates, with T. rubrum representing 64.29 %, T. interdigitale (T. mentagrophytes) (21.43 %) and Microsporum gypseum (14.29 %).

Pycard and BC017158 Candidate Genes of Irm1 Locus Modulate Inflammasome Activation for IL-1ß Production.

We identified Pycard and BC017158 genes as putative effectors of the Quantitative Trait locus (QTL) that we mapped at distal chromosome 7 named Irm1 for Inflammatory response modulator 1, controlling acute inflammatory response (AIR) and the production of IL-1ß, dependent on the activation of the NLRP3 inflammasome. We obtained the mapping through genome-wide linkage analysis of Single Nucleotide Polymorphisms (SNPs) in a cross between High (AIRmax) and Low (AIRmin) responder mouse lines that we produced by several generations of bidirectional selection for Acute Inflammatory Response. A highly significant linkage signal (LOD score peak of 72) for ex vivo IL-1ß production limited a 4 Mbp interval to chromosome 7. Sequencing of the locus region revealed 14 SNPs between "High" and "Low" responders that narrowed the locus to a 420 Kb interval. Variants were detected in non-coding regions of Itgam, Rgs10 and BC017158 genes and at the first exon of Pycard gene, resulting in an E19K substitution in the protein ASC (apoptosis associated speck-like protein containing a CARD) an adaptor molecule in the inflammasome complex. Silencing of BC017158 inhibited IL1-ß production by stimulated macrophages and the E19K ASC mutation carried by AIRmin mice impaired the ex vivo IL-1ß response and the formation of ASC specks in stimulated cells. IL-1ß and ASC specks play major roles in inflammatory reactions and in inflammation-related diseases. Our results delineate a novel genetic factor and a molecular mechanism affecting the acute inflammatory response.

Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs.

BACKGROUND: Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatile toxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clotting proteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to the structural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains, which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 of them belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and post-translational modifications. RESULTS: Thirty-one SVMP cDNAs were full length cloned from a single specimen of Bothrops neuwiedi snake, sequenced and grouped in eleven distinct sequences and further analyzed by cladistic analysis. Class P-I and class P-III sequences presented the expected tree topology for fibrinolytic and hemorrhagic SVMPs, respectively. In opposition, three distinct segregations were observed for class P-II sequences. P-IIb showed the typical segregation of class P-II SVMPs. However, P-IIa grouped with class P-I cDNAs presenting a 100% identity in the 365 bp at their 5' ends, suggesting post-transcription events for interclass recombination. In addition, catalytic domain of P-IIx sequences segregated with non-hemorrhagic class P-III SVMPs while their disintegrin domain grouped with other class P-II disintegrin domains suggesting independent evolution of catalytic and disintegrin domains. Complementary regions within cDNA sequences were noted and may participate in recombination either at DNA or RNA levels. Proteins predicted by these cDNAs show the main features of the correspondent classes of SVMP, but P-IIb and P-IIx included two additional cysteines cysteines at the C-termini of the disintegrin domains in positions not yet described. CONCLUSIONS: In B. neuwiedi venom gland, class P-II SVMPs were represented by three different types of transcripts that may have arisen by interclass recombination with P-I and P-III sequences after the divergence of the different classes of SVMPs. Our observations indicate that exon shuffling or post-transcriptional mechanisms may be driving these recombinations generating new functional possibilities for this complex group of snake toxins.

Influence of the expression vector and its elements on recombinant human prolactin synthesis in Escherichia coli; co-directional orientation of replication and transcription is highly critical.

The aim of the present work was to define a bacterial expression system that is particularly efficient for the synthesis of recombinant human prolactin (hPRL). In previous work, based on experiments that were basically carried out in parallel with the present ones, the synthesis of rec-hPRL by the p1813-hPRL vector in E. coli HB2151 was >500 mg/L, while it was much lower here (2.5-4-fold), in the RB791 and RRI strains. The highest positive influence on rec-hPRL synthesis was due to the transcription-replication co-orientation of hPRL cDNA and the ori/antibiotic resistance gene, responsible for up to a ~ 5-6-fold higher expression yield. In conclusion, this work confirmed that each bacterial strain of E. coli has a genetic background that can allow a different level of heterologous protein synthesis. The individual study of each element indicated that its action critically depends on the reading orientation in which it is located inside the vector: co-directional orientation of replication and transcription, in fact, greatly increased the level of rec-hPRL expression.

Update on human papilloma virus - part I: epidemiology, pathogenesis, and clinical spectrum.

Infection with human papilloma virus (HPV) is related to a great number of cutaneous and mucosal manifestations. The spectrum of HPV ranges from inapparent infections, through various clinical benign presentations including cutaneous and mucosal disease, to malignant and premalignant conditions. New HPV types are currently described in the literature; many of them are characterized as high-risk types due to their oncogenic potential. Knowledge regarding their epidemiology and pathogenesis is important to understand not only infection and disease processes, but also to formulate the clinical and laboratory basis for diagnosis, therapeutics, and prophylactic measures. This non-systematic review aims to discuss and to update those aspects, with an emphasis on relevant topics for dermatologists. HPV infection and related diseases in the Brazilian scenario are highlighted, including common dermatologic conditions seen at clinics as well as the condition of a public health problem as a sexually transmitted infection. The oncogenicity of the virus and the variety of clinical outcomes - especially in the immunocompromised individuals - are addressed.

Update on human papillomavirus - Part II: complementary diagnosis, treatment and prophylaxis.

In this nonsystematic review, the complementary diagnosis, treatment, prevention, and control of human papillomavirus are discussed. The histopathology is addressed regarding its indications, main findings and limitations, as a complementary diagnostic method largely used by dermatologists. Electron microscopy is briefly reviewed, along with its contribution to the accumulated knowledge on HPV, as well as the relevance of research in using this technology for future advances in diagnosis and treatment. Molecular information about the virus is continuously increasing, and the practical applications of HPV serology, molecular identification and genotyping are discussed. Vaccines are a valuable tool in primary HPV infection prevention and are now available in many countries; their composition, indications, and adverse effects are revisited. Local and systemic treatment options are reviewed and off-label prescriptions are discussed. Finally, health education focusing on HPV infection as a sexually transmitted infection of worldwide relevance and the many barriers to improve primary and secondary prevention are addressed.

Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing.

Human BMP-2, a homodimeric protein that belongs to the TGF- ß family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

Recombinant Production and Characterization of a New Toxin from Cryptops iheringi Centipede Venom Revealed by Proteome and Transcriptome Analysis.

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work.

Human bone morphogenetic protein-2 (hBMP-2) characterization by physical-chemical, immunological and biological assays.

Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor ß (TGF-ß) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, size-exclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived met-hBMP-2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts.

Design and Production of a Recombinant Hybrid Toxin to Raise Protective Antibodies Against Loxosceles Spider Venom.

Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen's formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.

When spider and snake get along: Fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell.

Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.

Case for diagnosis. Patch granuloma annulare.

A 59-year-old woman reported a 20-day history of slightly scaly erythematous infiltrated patches on her palms and soles with a histopathological result which was consistent with interstitial-pattern granuloma annulare, clinically classified as patch granuloma annulare. This is a rare clinical variant of granuloma annulare, with an unknown incidence and characteristic clinical and histopathological features. The patient evolved with a complete remission of the lesions after biopsy and the use of high-potency topical corticosteroid.

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria.

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

Recombinant Phospholipase D from Loxosceles gaucho Binds to Platelets and Promotes Phosphatidylserine Exposure.

Spider envenomation, from the genus Loxosceles, is frequently reported as a cause of necrotic lesions in humans around the world. Among the many components found in the venom of Loxosceles genus, phospholipases D (PLDs) are the most investigated, since they can cause a massive inflammatory response, dermonecrosis, hemolysis and platelet aggregation, among other effects. Even though the PLDs induce strong platelet aggregation, there are no studies showing how the PLDs interact with platelets to promote this effect. Since many agonists must interact with specific receptors on the platelet membrane to induce aggregation, it is reasonable to expect that the PLDs may, in some way, also interact with platelets, to induce this activity. Therefore, to address this possibility, in this work, a recombinant PLD, called LgRec1, from L. gaucho was fused to enhanced green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 to platelets, by fluorescence-activated cell sorter (FACS) and confocal microscopy. The preservation of biological activities of this chimera toxin was also analyzed. As a first, the results show that LgRec1 does not require plasma components to bind to platelets, although these components are necessary to LgRec1 to induce platelet aggregation. Also, the attachment of LgRec1 to human platelets' cell membranes suggests that the exposure of phosphatidylserine (PS) may act as a scaffold for coagulation factors. Therefore, the results add new information about the binding of Loxosceles PLDs to platelets, which may help unravel how these toxins promote platelet aggregation.

Psoriasis and pentoxifylline: a clinical, histopathologic, and immunohistochemical evaluation.

OBJECTIVE: The aim of this study was to examine the clinical and histopathologic effects of pentoxifylline on psoriasis, compared with placebo. METHODS: Sixty-one outpatients with active psoriasis were randomly assigned to either of 2 groups: one was given pentoxifylline 400 mg tid PO, and the other, placebo. Fifty-six patients concluded the study. They were evaluated clinically and by laboratory parameters before and after 8 weeks of treatment. Pretreatment and posttreatment biopsies were taken. Initial sections were stained with hematoxylin-eosin. Further cuts were immunostained for cytokeratins 10, 14, and 16. RESULTS: Clinical and histologic improvement did not show statistically significant differences between the groups. No laboratory abnormalities or serious reactions related to the drug were observed. CONCLUSIONS: No statistical difference was seen when the treatment group was compared with the control group. Pentoxifylline is a well tolerated and safe drug, but its efficacy in psoriasis appears to be limited.

A novel human G protein-coupled receptor is over-expressed in prostate cancer.

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological functions. The number of known members that belong to this large family of receptors has been rapidly increasing. Now, with the availability of the human genome sequence databases, further family members are being identified. We describe the identification of a novel GPCR that shows no significant amino acid identity to any one of the known members of the GPCR superfamily. The gene expression pattern of this receptor is restricted: in normal tissues it is confined to the nervous system and testis, but we also detected gene expression in several tumor types, most notably prostate cancer, suggesting a potential role for this gene as a marker for this disease.