A Mini-Review of Diagnostic Methods for the Antigen and Antibody Detection of Rocky Mountain and Brazilian Spotted Fever.

Rocky Mountain or Brazilian spotted fever, caused by Rickettsia rickettsii, is a fulminant, seasonal, and neglected disease that occurs in focal points of North America and South America. Its rapid detection is essential for the better prognosis and survival rate of infected individuals. However, disease diagnosis still faces challenges as the accuracy of many of the available laboratory tests fluctuates. This review aimed to analyze methods for antibody or antigen detection, their gaps, and their evolution over time. A search was conducted to find all studies in the Pubmed database that described the antibody or antigen detection of R. rickettsii infections. Initially, a total of 403 articles were screened. Of these articles, only 17 fulfilled the pre-established inclusion criteria and were selected. Among the different methods applied, the IFA technique was the one most frequently found in the studies. However, it presented varied results such as a low specificity when using the indirect method. Other techniques, such as ELISA and immunohistochemistry, were also found, although in smaller numbers and with their own limitations. Although some studies showed promising results, there is a pressing need to find new techniques to develop a rapid and effective diagnosis of R. rickettssi infection.

Biodistribution and Tumor Targeted Accumulation of Anti-CEA-loaded Iron Nanoparticles.

INTRODUCTION: Active targeting of tumors by nanomaterials favors early diagnosis and the reduction of harsh side effects of chemotherapeuticals. METHOD: We synthesized magnetic nanoparticles (64 nm; -40 mV) suspended in a magnetic fluid (MF) and decorated them with anti-carcinoembryonic antigen (MFCEA; 144 nm; -39 mV). MF and MFCEA nanoparticles were successfully radiolabeled with technetium-99m (99mTc) and intravenously injected in CEA-positive 4T1 tumor-bearing mice to perform biodistribution studies. Both 99mTc-MF and 99mTc-MFCEA had marked uptake by the liver and spleen, and the renal uptake of 99mTc-MFCEA was higher than that observed for 99mTc-MF at 20h. At 1 and 5 hours, the urinary excretion was higher for 99mTc-MF than for 99mTc-MFCEA. RESULTS: These data suggest that anti-CEA decoration might be responsible for a delay in renal clearance. Regarding the tumor, 99mTc-MFCEA showed tumor uptake nearly two times higher than that observed for 99mTc-MFCEA. Similarly, the target-nontarget ratio was higher with 99mTc-MFCEA when compared to the group that received the 99mTc-MF. CONCLUSION: These data validated the ability of active tumor targeting by the as-developed antiCEA loaded nanoparticles and are very promising results for the future development of a nanodevice for the management of breast cancer and other types of CEA-positive tumors.

A mini-review on ELISA-Based Diagnosis of Schistosomiasis.

BACKGROUND: schistosomiasis is a neglected tropical parasitic disease caused by trematode worms of the genus schistosoma, which affects approximately 240 million people worldwide. the diagnosis of the disease can be performed by parasitological, molecular, and/or immunological methods, however, the development of new diagnostic methods still essential to guide policy decisions, monitor disease trends and assess the effectiveness of interventions. OBJECTIVE: in this sense, the current work summarizes the findings of a systematic review regarding antigens applied in the enzyme-linked immunosorbent assay test, which were patented and published over the last ten years. METHODS: the literature search strategy used medical subject heading (mesh) terms to define as descriptors. "schistosoma mansoni" was used in arrangement with the descriptors "immunoassay", "enzyme-linked immunosorbent assay", "elisa", and "antigens", using the "and" connector. the patent search was done using keywords, including diagnosis and schistosoma or schistosomiasis or schistosome. several databases were employed for the patent search, such as intellectual property national institute; european patent office; the united states patent and trademark office; patent scope, and google patents. RESULTS: forty-one articles were retrieved, of which only five met the eligibility criteria. seventeen patents were taken from the databases, and a brief description of the most relevant inventions is given here. CONCLUSION: schistosomiasis is considered the most important helminthic disease in worldwide. therefore, it is important to of searching for and develops diagnostic methods based on serology to reduce morbidity and mortality caused by the disease.

A Mini-review on Potentials Proteins/Peptides Applied for Serodiagnosis of Human Monkeypox Infection and Future Trends.

Monkeypox is a zoonosis that re-emerged in 2022, generating cases in non-endemic countries for the disease and creating a public health issue. The rapid increase in the number of cases kindles a need for quick, inexpensive diagnostic tests for the epidemiological control of the disease. The high cost of molecular tests can make this control more difficult to access in poorer regions, with immunological tests being a more viable option. In this mini-review, a search was conducted in the main databases for peptide and protein options that could be used in the development of serological diagnostic tests. Nine viable registres were found, and seven were selected (two patents and five studies). The main studies used the B21R peptide sequence as it is a high immunogenic epitope. In addition, studies on the improvement of these sequences were also found to avoid cross-reactions against other viruses of the same family, proposing a rational approach using multiepitope recombinant proteins. These approaches demonstrated high sensitivity and specificity values and are seen as viable options for developing new tests. New effective serological testing options, when combined with awareness, disease surveillance, early diagnosis, and rapid communication, form a set of key strategies used by health systems to control the spread of the monkeypox virus.

rMELEISH: A Novel Recombinant Multiepitope-Based Protein Applied to the Serodiagnosis of Both Canine and Human Visceral Leishmaniasis.

BACKGROUND: visceral leishmaniasis (VL) is a critical public health problem in over ninety countries. The control measures adopted in Brazil have been insufficient when it comes to preventing the spread of this overlooked disease. In this context, a precise diagnosis of VL in dogs and humans could help to reduce the number of cases of this disease. Distinct studies for the diagnosis of VL have used single recombinant proteins in serological assays; however, the results have been variable, mainly in relation to the sensitivity of the antigens. In this context, the development of multiepitope-based proteins could be relevant to solving such problem. METHODS: a chimeric protein (rMELEISH) was constructed based on amino acid sequences from kinesin 39 (k39), alpha-tubulin, and heat-shock proteins HSP70 and HSP 83.1, and tested in enzyme-linked immunosorbent (ELISA) for the detection of L. infantum infection using canine (n = 140) and human (n = 145) sera samples. RESULTS: in the trials, rMELEISH was able to discriminate between VL cases and cross-reactive diseases and healthy samples, with sensitivity and specificity values of 100%, as compared to the use of a soluble Leishmania antigenic extract (SLA). CONCLUSIONS: the preliminary data suggest that rMELEISH has the potential to be tested in future studies against a larger serological panel and in field conditions for the diagnosis of canine and human VL.

Proof of Concept of a Novel Multiepitope Recombinant Protein for the Serodiagnosis of Patients with Chagas Disease.

Chagas disease remains a neglected disease that is considered to be a public health problem. The early diagnosis of cases is important to improve the prognosis of infected patients and prevent transmission. Serological tests are the method of choice for diagnosis. However, two serological tests are currently recommended to confirm positive cases. In this sense, more sensitive and specific serological tests need to be developed to overcome these current diagnosis problems. This study aimed to develop a new recombinant multiepitope protein for the diagnosis of Chagas disease, hereafter named rTC. The rTC was constructed based on amino acid sequences from different combinations of Trypanosoma cruzi antigens in the same polypeptide and tested using an enzyme-linked immunosorbent assay (ELISA) to detect different types of Chagas disease. rTC was able to discriminate between indeterminate (IND) and cardiac (CARD) cases and cross-reactive diseases, as well as healthy samples, with 98.28% sensitivity and 96.67% specificity, respectively. These data suggest that rTC has the potential to be tested in future studies against a larger serological panel for the diagnosis of Chagas disease.

A Biotechnological Review on Patents Applied to Rubella Diagnosis.

BACKGROUND: Rubella, caused by the Rubella virus (RV), is considered a mild self-limited illness. However, RV has teratogenic potential. Laboratory investigation plays an important role in both diagnosis and surveillance of the disease. The main methods for diagnosing Rubella are serological assays for the detection of specific IgM and molecular assays for detecting viral RNA. However, some laboratories perform IgG avidity testing, virus isolation and analysis of genetic sequence as tools to help Rubella eradication. The importance of the diagnosis of Rubella involves the appropriate treatment of the disease, because the Rubella clinical symptoms may be similar to those of other diseases, and the population monitoring to avoid new emergent cases. This study addresses different methods of diagnosing Rubella and contributes as a source of knowledge to assist health systems in controlling the disease. OBJECTIVE: The main objective of this study was to review the available patents regarding Rubella diagnosis published in intellectual property databases, and provides an overview of the technologies available for the diagnosis of Rubella. METHOD: The search strategy was based on the keywords searched separately or together using a Boolean operator either in the patent title or abstract the time interval was restricted to patents filed or granted from January 2009 until February 2022. The database used was Google Patents. RESULTS: This study analyzed 24 patent documents regarding strategies for the diagnosis of Rubella. Of these, 15 patents disclose strategies for detecting Rubella antibodies, 7 patents the detection of Rubella virus nucleic acid, and 2 patents the production of antibodies applied in Rubella diagnosis. CONCLUSION: Rubella is still a public health problem in some countries, mainly those in development, especially due to congenital Rubella syndrome, which can cause malformation or fetal death. However, its diagnosis is challenging, due to similarity of symptoms with other diseases, and for this reason, laboratory diagnosis is essential. Studies like this encourage researchers and governments to invest in research to continue the development of new products, using different areas of biotechnology, to solve society's problems, especially diseases that have an impact on global health, such as Rubella.

Anti-CEA tagged iron nanoparticles for targeting triple-negative breast cancer.

Systemic therapy is generally required for breast cancer. However, treatment toxicity and side effects are a concern, especially for triple-negative breast cancer (TNBC), a subtype that usually develops resistance to chemotherapy. To overcome this issue, new nanoformulations capable of targeting cancer cells have been developed and alternative biomarkers have been explored as target molecules for TNBC management. In this study, we performed anin vivoassay in a murine orthotopic TNBC model to evaluate the targeting ability of anti-carcinoembryonic antigen (anti-CEA) loaded nanoparticles (labelled MFCEA), which had been previously synthetized by our research group. 4T1 cells were injected in the mammary gland of balb-c mice, and tumors were evaluated for CEA expression by immunohistochemistry. Tumor-bearing mice received targeted (MFCEA) and non-targeted (MF) nanoparticles intraperitoneally. Tumors were removed 1, 4, 15 and 24 h after treatment, and Prussian blue iron staining was performed. Our results showed, as far as we know for the first time, that 4T1 induced tumors are CEA positive, and this opens up new prospects for treating TNBC. Furthermore, MFCEA nanoparticles were able to target malignant tissue and were retained in the tumor for longer than MF nanoparticles. The retention property of MFCEA, together with the absence of toxicity observed in the MTT assay, make these nanoparticles a promising device for management of CEA positive tumors and perhaps for TNBC. Nevertheless, further studies must be carried out to improve their performance and ensure safety for clinical studies.

Diagnostic markers selected by immunoproteomics and phage display applied for the serodiagnosis of canine leishmaniosis.

Canine leishmaniosis (CanL) is one of the most important parasitic diseases found in several countries worldwide. Dogs are considered important domestic reservoirs of the parasites, being relevant in the maintenance of transmission cycle of the disease between sandflies and humans. However, the prevalence of asymptomatic infection is considerably higher than that of apparent clinical illness in the infected animals; thus making promptly necessary to diagnose the infection in these animals, which could help to allow to the adoption of more efficient control measures against disease. Parasitological tests, which are considered as gold standard to demonstrate the infection and diagnose the disease, present problems related with their sensitivity. Also, the sample´s collect is considered invasive. As consequence, serological tests could be applied as an additional tool to detect the asymptomatic and symptomatic CanL. For this purpose, distinct recombinant antigens have been studied; however, problems in their sensitivity and/or specificity have been still registered. The present review focus in advances in the identification of new diagnostic targets applied for the CanL diagnose, represented here by recombinant single, combined or chimeric proteins, as well as by peptides that mimic epitopes (mimotopes); which were selected by means of immunoproteomics and phage display.

A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis.

BACKGROUND: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. OBJECTIVE: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. METHODS: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). RESULTS: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. CONCLUSION: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

Expression and Biochemical Characterization of a Yersinia intermedia Phytase Expressed in Escherichia coli.

BACKGROUND: Phytases are enzymes capable of degrading phytic acid and used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. OBJECTIVE: The main goal of this study was to express and characterize a Yersinia intermedia phytase expressed in Escherichia coli cells. METHODS: The Y. intermedia phytase gene was synthesized and overexpressed in Escherichia coli cells. The phytase recombinante (rPHY) was purified to homogeneity using a Ni-NTA column. The biochemical and biophysical properties of the rPHY were measured in order to fully characterize the recombinant enzyme. The following patents database were consulted: Espacenet, USPTO, LATIPAT, Patent Scope, WIPO and Google Patents. RESULTS: The results showed that the rPHY is active at 37-40ºC and presented an optimal pH and temperature of 8.0 and 40°C, respectively. The phytase rPHY was activated by Cu2+ ion and showed resistance to trypsin and pepsin, retaining 55% of the activity at the ratio of 0.02. Furthermore, the dissociation constant (Kd = 1.1150 ± 0.0087 mM), as estimated by a fluorescence binding assay, suggests a medium affinity of the enzyme with the substrate. CONCLUSION: The results of this article can be considered as innovative and for this reason, they were protected by Intellectual Property Law in Brazil. Take together, the biochemical properties of the rPHY could be useful in future for its industrial application of this enzyme as an additive in the monogastric feed.

Carcinoembryonic Antigen (CEA) and Hepatic Metastasis in Colorectal Cancer: Update on Biomarker for Clinical and Biotechnological Approaches.

BACKGROUND: Carcinoembryonic Antigen (CEA) is a recommended prognostic marker in Colorectal Cancer (CRC) for tumor diagnosis and monitoring response to therapy. High CEA levels are specifically associated with CRC progression and increased levels of the marker are expected to fall following surgical treatment. Due to its role in CRC, CEA has also been explored as a target for cancer therapy and diagnosis approaches. OBJECTIVE: The goal of this work is to highlight the role of CEA in CRC progression and liver metastasis as well as its potential as a biomarker for clinical and biotechnological approaches. METHOD: A literature search of electronic medical and patent databases Pubmed, Scopus, and Science Direct, Google patents, Esp@cenet and United States Patent and Trademark Office (USPTO), was performed. Information was collected in recent publications, including 81 articles besides 13 patents related to different CEA targeting biotechnological approaches for CRC therapy and diagnosis. RESULTS: CEA enhances CRC metastatic potential through many ways. CEA protects metastatic cells from death, changes the microenvironment of sinusoids, promoting the expression of adhesion molecule and malignant cell survival, besides being considered a proangiogenic molecule. Furthermore CEA has also been evaluated as a target in drug delivery systems, photodynamic therapy, radioimmunotherapy, cancer imaging and nanotechnological devices, leading to many patents concerning to development of anti-CEA antibodies or their fragments with potential to target colorectal cancer and liver metastasis cells. CONCLUSION: CEA is already clinically used to monitor CRC patients, and it is a very promising targeting biomarker for multiple biotechnological applications. As far as we know this is the first report on CEA that addresses patents database.

Biofactories for the Production of Recombinant Phytases and their Application in the Animal Feed Industry.

BACKGROUND: Phytases are enzymes capable of degrading phytic acid and are used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. Recent inventions show interest in production and optimization of recombinant phytases with biochemical and physicochemical characteristics promising for animal feed industry. METHODS: This review article is focused on relevant patents of promising phytases, together with the commonly used expression systems for their production and tools currently employed to generate new phytases. We revised all patents related to recombinant phytases and their application in the animal feed industry. The following patents databases were consulted: European Patent Office (Espacenet), the United States Patent and Trademark Office (USPTO), the United States Latin America Patents (LATIPAT), Patent scope -Search International and National Patent Collections (WIPO) and Google Patents. RESULTS: In this review, information was collected from recent publications, including 38 patents related production systems for different recombinant phytases and their application in the animal feed industry. CONCLUSION: We showed that important recombinant phytases were successfully produced in different expression systems. In addition, this work highlights certain biotechnological tools such as mutagenesis for generation of novel enzymes with biochemical properties of use in the animal feed industry.

Cytotoxicity of Portland cement with different radiopacifying agents: a cell death study.

INTRODUCTION: The aim of this study was to investigate the cytotoxicity of white Portland cement (PC) alone or associated with bismuth oxide (PCBi), zirconium oxide (PCZir), and calcium tungstate (PCCa) in 2 cell lineages. METHODS: Murine periodontal ligament cells (mPDL) and rat osteosarcoma cells (ROS 17/2.8) were exposed for 24 hours to specific concentrations of fresh PC and PC associations with radiopacifiers. Zinc oxide-eugenol cement and hydrogen peroxide treatment were applied as cytotoxic positive controls. Cell viability after incubation with the cements was assessed by mitochondrial dehydrogenase enzymatic assay. Cell morphology was microscopically analyzed by cresyl violet staining, and the mechanism of cell death was determined by acridine orange/ethidium bromide methodology. All data were analyzed statistically by analysis of variance and Tukey post hoc test (P < .05). The correlation among cell death by apoptosis or necrosis and pH values was established by Pearson linear coefficient. RESULTS: The mitochondrial dehydrogenase enzymatic assay only revealed significant cell death rate at high concentrations of cement elutes. PC alone was not cytotoxic, even at 100 mg/mL. Microscopic images showed that none of the PC formulations caused damage to any cell lines. Statistical analysis of apoptosis/necrosis data demonstrated that PC and PC plus radiopacifying agents promoted significant necrosis cell death only at 100 mg/mL. CONCLUSIONS: The mPDL cells were more sensitive than ROS17/2.8. The results showed that PC associated with bismuth oxide, zirconium oxide, or calcium tungstate is not cytotoxic to mPDL or ROS17/2.8. Zirconium oxide and calcium tungstate might be good alternatives as radiopacifying agents.

Interaction of bracken-fern extract with vitamin C in human submandibular gland and oral epithelium cell lines.

The consumption of bracken-fern (Pteridium aquilinum) as food is associated with a high incidence of cancer in humans and animals. Thus far, the carcinogenic effects of bracken-fern consumption could be related to chromosome aberrations verified in animal and in human peripheral lymphocytes. We tested the in vitro effects of vitamin C (10 and 100 microg/ml) on the reversibility of DNA damage caused by bracken-fern on human submandibular gland (HSG) cells and on oral epithelium cells (OSCC-3) previously exposed to bracken-fern extract. DNA damage (i.e. nuclei with increased levels of DNA migration) was determined by comet assay, cell morphology was evaluated by light microscopy and cellular degeneration was assessed by the acridine orange/ethidium bromide fluorescent-dyeing test. Results showed that vitamin C alone did not reduce DNA damage caused by bracken-fern in HSG and OSSC-3 cells. However, at a higher concentration (100 microg/ml), vitamin C induced DNA damage in both cell lines. Moreover, vitamin C (10 and 100 microg/ml) together with bracken-fern extract showed synergistic effects on the frequency of DNA damage in HSG cells. In addition, cells treated with bracken-fern extract or vitamin C alone, or with their association, showed apoptosis morphological features, such as chromatin condensation, cytoplasmic volume loss, changes in membrane symmetry and the appearance of vacuoles; these alterations were observed in both cell lines. These results demonstrate that bracken-fern extract was cytotoxic to HSG and OSCC-3 cells, causing cell death by apoptosis, and that vitamin was not able to revert these effects.

Production of recombinant human factor VIII in different cell lines and the effect of human XBP1 co-expression.

Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.