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1.
EBioMedicine ; 46: 215-226, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31326432

RESUMO

BACKGROUND: Profiles of immunity developed in filovirus patients and survivors have begun to shed light on antigen-specific cellular immune responses that had been previously under-studied. However, our knowledge of the breadth and length of those responses and the viral targets which mediate long-term memory immunity still lags significantly behind. METHODS: We characterized antigen-specific immune responses in whole blood samples of fifteen years post-infected survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). We examined T cell and IgG responses against SUDV complete antigen and four SUDV proteins; glycoprotein (GP), nucleoprotein (NP), and viral protein 30 (VP30), and 40 (VP40). FINDINGS: We found survivors-maintained antigen-specific CD4+ T cell memory immune responses mediated mainly by the viral protein NP. In contrast, activated CD8+ T cell responses were nearly absent in SUDV survivors, regardless of the stimulating antigen used. Analysis of anti-viral humoral immunity revealed antigen-specific IgG antibodies against SUDV and SUDV proteins. Survivor IgGs mediated live SUDV neutralization in vitro and FcγRI and FcγRIII antibody Fc-dependent responses, mainly via antibodies to the viral proteins GP and VP40. INTERPRETATION: We highlight the key role of several proteins, i.e., GP, NP, and VP40, to act as mediators of distinctive and sustained cellular memory immune responses in long-term SUDV survivors. We suggest that the inclusion of these viral proteins in vaccine development may best mimic survivor native memory immune responses with the potential of protecting against viral infection. FUNDS: This research was funded by the Defense Threat Reduction Agency (CB4088) and by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI111516. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

2.
Transbound Emerg Dis ; 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31127983

RESUMO

Here, we report the results of a cross-sectional study designed to monitor the circulation and genetic diversity of foot and mouth disease virus (FMDV) in Uganda between 2014 and 2017. In this study, 13,614 sera and 2,068 oral-pharyngeal fluid samples were collected from cattle and analysed to determine FMDV seroprevalence, circulating serotypes and their phylogenetic relationships. Circulation of FMDV was evidenced by the detection of antibodies against non-structural proteins of FMDV or viral isolations in all districts sampled in Uganda. Sequence analysis revealed the presence of FMDV serotypes A, O, SAT 1 and SAT 2. FMDVs belonging to serotype O, isolated from 21 districts, were the most prevalent and were classified into six lineages within two East African topotypes, namely EA-1 and EA-2. Serotype A viruses belonging to the Africa G-I topotype were isolated from two districts. SAT 1 viruses grouped within topotypes I and IV and SAT 2 viruses within topotypes VII, IV and X were isolated from six and four districts respectively. Phylogenetic analysis of SAT 1 and SAT 2 sequences from cattle clustered with historical sequences from African buffalo, indicating possible interspecies transmission at the wildlife-livestock interface. In some cases, Uganda viruses also shared similarities to viral strains recovered from other regions in East Africa. This 3-year study period provides knowledge about the geographical distribution of FMDV serotypes isolated in Uganda and insights into the genetic diversity of the multiple serotypes circulating in the country. Knowledge of circulating FMDV viruses will assist in antigenic matching studies to devise improved FMDV control strategies with vaccination and vaccine strain selection for Uganda.

4.
Front Vet Sci ; 5: 250, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30370272

RESUMO

Effective management of foot and mouth disease (FMD) requires diagnostic tests to distinguish between infected and vaccinated animals (DIVA). To address this need, several enzyme-linked immunosorbent assay (ELISA) platforms have been developed, however, these tests vary in their sensitivity and specificity and are very expensive for developing countries. Camelid-derived single-domain antibodies fragments so-called Nanobodies, have demonstrated great efficacy for the development of serological diagnostics. This study describes the development of a novel Nanobody-based FMD 3ABC competitive ELISA, for the serological detection of antibodies against FMD Non-Structural Proteins (NSP) in Uganda cattle herds. This in-house ELISA was validated using more than 600 sera from different Uganda districts, and virus serotype specificities. The evaluation of the performance of the assay demonstrated high diagnostic sensitivity and specificity of 94 % (95 % CI: 88.9-97.2), and 97.67 % (95 % CI: 94.15-99.36) respectively, as well as the capability to detect NSP-specific antibodies against multiple FMD serotype infections. In comparison with the commercial PrioCHECK FMDV NSP-FMD test, there was a strong concordance and high correlation and agreement in the performance of the two tests. This new developed Nanobody based FMD 3ABC competitive ELISA could clearly benefit routine disease diagnosis, the establishment of disease-free zones, and the improvement of FMD management and control in endemically complex environments, such as those found in Africa.

5.
Front Immunol ; 9: 1428, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30013549

RESUMO

The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.

6.
Front Immunol ; 9: 1025, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867992

RESUMO

The recent West African Ebola virus pandemic, which affected >28,000 individuals increased interest in anti-Ebolavirus vaccination programs. Here, we systematically analyzed the requirements for a prophylactic vaccination program based on the basic reproductive number (R0, i.e., the number of secondary cases that result from an individual infection). Published R0 values were determined by systematic literature research and ranged from 0.37 to 20. R0s ≥ 4 realistically reflected the critical early outbreak phases and superspreading events. Based on the R0, the herd immunity threshold (Ic) was calculated using the equation Ic = 1 - (1/R0). The critical vaccination coverage (Vc) needed to provide herd immunity was determined by including the vaccine effectiveness (E) using the equation Vc = Ic/E. At an R0 of 4, the Ic is 75% and at an E of 90%, more than 80% of a population need to be vaccinated to establish herd immunity. Such vaccination rates are currently unrealistic because of resistance against vaccinations, financial/logistical challenges, and a lack of vaccines that provide long-term protection against all human-pathogenic Ebolaviruses. Hence, outbreak management will for the foreseeable future depend on surveillance and case isolation. Clinical vaccine candidates are only available for Ebola viruses. Their use will need to be focused on health-care workers, potentially in combination with ring vaccination approaches.

7.
ACS Nano ; 12(1): 63-73, 2018 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-29303554

RESUMO

Ebola virus disease causes widespread and highly fatal epidemics in human populations. Today, there is still great need for point-of-care tests for diagnosis, patient management and surveillance, both during and post outbreaks. We present a point-of-care test comprising an immunochromatographic strip and a smartphone reader, which detects and semiquantifies Ebola-specific antibodies in human survivors. We developed a Sudan virus glycoprotein monoplex platform and validated it using sera from 90 human survivors and 31 local noninfected controls. The performance of the glycoprotein monoplex was 100% sensitivity and 98% specificity compared to standard whole antigen enzyme-linked immunosorbent assay (ELISA), and it was validated with freshly collected patient samples in Uganda. Moreover, we constructed a multiplex test for simultaneous detection of antibodies against three recombinant Sudan virus proteins. A pilot study comprising 15 survivors and 5 noninfected controls demonstrated sensitivity and specificity of 100% compared to standard ELISA. Finally, we developed a second multiplex subtype assay for the identification of exposure to three related EVD species: Sudan virus, Bundibugyo virus and Ebola virus (formerly Zaire) using recombinant viral glycoprotein. This multiplex test could distinguish between the host's immunity to specific viral species and identify cross-reactive immunity. These developed serological platforms consisted of capture ligands with high specificity and sensitivity, in-house developed strips and a compatible smartphone application. These platforms enabled rapid and portable testing, data storage and sharing as well as geographical tagging of the tested individuals in Uganda. This platform holds great potential as a field tool for diagnosis, vaccine development, and therapeutic evaluation.

8.
Sci Rep ; 7(1): 6054, 2017 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729706

RESUMO

Ebolavirus is a highly lethal pathogen, causing a severe hemorrhagic disease with a high fatality rate. To better understand immune correlates of protection by virus specific IgG, we investigated the evolution of the Fcγ receptors (FcγRs)-activating capabilities of antiviral IgG in serum samples of long recovered survivors. To this end, longitudinal serum samples from survivors of Sudan ebolavirus (SUDV) infection, studied over years, were examined for the presence of Ebola-GP specific IgG subclasses, and for their binding to FcγRs. We developed a cell-based reporter system to quantitate pathogen-specific antibody binding to FcγRIIIA, FcγRIIA, FcγRIIB and FcγRI. With this system, we demonstrate that anti-GP-specific stimulation of the FcγRI reporter by survivors' sera was substantially high one year after acute infection, with a slight reduction in activity over a decade post infection. We further demonstrate that GP-specific IgG1 is by far the seroprevalent subclass that retained and even enhanced its presence in the sera, over ten years post infection; the prevalence of other GP-specific IgG subclasses was considerably reduced over time. In accordance, GP-specific FcγRI reporter response and GP-specific total IgG1 subclass correlated in the studied group of Ebola survivors. These observations are important for further informing Ebola vaccine and therapeutic development.

9.
J Exp Med ; 214(9): 2563-2572, 2017 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-28724616

RESUMO

Until recently, immune responses in filovirus survivors remained poorly understood. Early studies revealed IgM and IgG responses to infection with various filoviruses, but recent outbreaks have greatly expanded our understanding of filovirus immune responses. Immune responses in survivors of Ebola virus (EBOV) and Sudan virus (SUDV) infections have provided the most insight, with T cell responses as well as detailed antibody responses having been characterized. Immune responses to Marburg virus (MARV), however, remain almost entirely uncharacterized. We report that immune responses in MARV survivors share characteristics with EBOV and SUDV infections but have some distinct differences. MARV survivors developed multivariate CD4+ T cell responses but limited CD8+ T cell responses, more in keeping with SUDV survivors than EBOV survivors. In stark contrast to SUDV survivors, rare neutralizing antibody responses in MARV survivors diminished rapidly after the outbreak. These results warrant serious consideration for any vaccine or therapeutic that seeks to be broadly protective, as different filoviruses may require different immune responses to achieve immunity.


Assuntos
Anticorpos Neutralizantes/imunologia , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Células Th1/imunologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunidade Celular/imunologia , Masculino , Doença do Vírus de Marburg/mortalidade , Pessoa de Meia-Idade , Sobreviventes , Uganda/epidemiologia , Adulto Jovem
10.
Clin Vaccine Immunol ; 23(8): 717-24, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27335383

RESUMO

A detailed understanding of serological immune responses to Ebola and Marburg virus infections will facilitate the development of effective diagnostic methods, therapeutics, and vaccines. We examined antibodies from Ebola or Marburg survivors 1 to 14 years after recovery from disease, by using a microarray that displayed recombinant nucleoprotein (NP), viral protein 40 (VP40), envelope glycoprotein (GP), and inactivated whole virions from six species of filoviruses. All three outbreak cohorts exhibited significant antibody responses to antigens from the original infecting species and a pattern of additional filoviruses that varied by outbreak. NP was the most cross-reactive antigen, while GP was the most specific. Antibodies from survivors of infections by Marburg marburgvirus (MARV) species were least cross-reactive, while those from survivors of infections by Sudan virus (SUDV) species exhibited the highest cross-reactivity. Based on results revealed by the protein microarray, persistent levels of antibodies to GP, NP, and VP40 were maintained for up to 14 years after infection, and survival of infection caused by one species imparted cross-reactive antibody responses to other filoviruses.


Assuntos
Anticorpos Antivirais/sangue , Reações Cruzadas , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Animais , Formação de Anticorpos , Antígenos Virais/imunologia , Estudos de Coortes , Surtos de Doenças , Voluntários Saudáveis , Doença pelo Vírus Ebola/epidemiologia , Humanos , Doença do Vírus de Marburg/epidemiologia , Análise em Microsséries , Análise Serial de Proteínas , Sobreviventes , Proteínas Estruturais Virais/genética
11.
Viruses ; 8(5)2016 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-27187443

RESUMO

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Testes de Neutralização , Sobreviventes , Uganda
12.
Viruses ; 7(1): 37-51, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25569078

RESUMO

Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda-Gulu 2000-2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1-649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1-649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.


Assuntos
Surtos de Doenças , Ebolavirus/imunologia , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/imunologia , Memória Imunológica , Anticorpos Antivirais/sangue , Estudos de Coortes , Reações Cruzadas , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunidade Celular , Imunoglobulina G/sangue , Testes de Neutralização , Sudão , Sobreviventes , Uganda/epidemiologia
14.
J Infect Dis ; 208(2): 299-309, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23585686

RESUMO

To better understand humoral immunity following ebolavirus infection, a serological study of the humoral immune response against the individual viral proteins of Sudan ebolavirus (Gulu) in human survivors was performed. An enzyme-linked immunosorbent assay specific for full-length recombinant viral proteins NP, VP30, VP40, and GP1-649 (GP lacking the transmembrane domain) of Sudan ebolavirus (Gulu) was used as well as a plaque reduction neutralization test. Serum samples from human survivors, which were collected up to 10 years following recovery, were screened and analyzed. Results demonstrate that samples obtained 10 years following infection contain virus-specific antibodies that can neutralize virus. Neutralization correlates well with immunoreactivity against the viral proteins NP, VP30, and GP1-649. Sera from individuals who died or those with no documented infection but immunoreactive to ebolavirus did not neutralize. This work provides insight into the duration, profile of immunoreactivity, and neutralization capacity of the humoral immune response in ebolavirus survivors.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Linhagem Celular , Cercopithecus aethiops , Células HEK293 , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Imunidade Humoral/imunologia , Imunoglobulina A/imunologia , Testes de Neutralização/métodos , Sudão , Sobreviventes , Células Vero , Proteínas do Envelope Viral/imunologia
15.
Clin Vaccine Immunol ; 19(11): 1844-52, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22993411

RESUMO

Ebolavirus, a member of the family Filoviridae, causes high lethality in humans and nonhuman primates. Research focused on protection and therapy for Ebola virus infection has investigated the potential role of antibodies. Recent evidence suggests that antibodies can be effective in protection from lethal challenge with Ebola virus in nonhuman primates. However, despite these encouraging results, studies have not yet determined the optimal antibodies and composition of an antibody cocktail, if required, which might serve as a highly effective and efficient prophylactic. To better understand optimal antibodies and their targets, which might be important for protection from Ebola virus infection, we sought to determine the profile of viral protein-specific antibodies generated during a natural cycle of infection in humans. To this end, we characterized the profile of antibodies against individual viral proteins of Sudan Ebola virus (Gulu) in human survivors and nonsurvivors of the outbreak in Gulu, Uganda, in 2000-2001. We developed a unique chemiluminescence enzyme-linked immunosorbent assay (ELISA) for this purpose based on the full-length recombinant viral proteins NP, VP30, and VP40 and two recombinant forms of the viral glycoprotein (GP(1-294) and GP(1-649)) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP(1-649), followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP(1-649), and VP40 demonstrated a high correlation of immunoreactivity between these viral proteins, which is also linked with survival. Overall, our studies of the profile of immunorecognition of antibodies against four viral proteins of Sudan Ebola virus in human survivors may facilitate development of effective monoclonal antibody cocktails in the future.


Assuntos
Anticorpos Antivirais/sangue , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Imunidade Humoral , Antígenos Virais/imunologia , Surtos de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Doença pelo Vírus Ebola/epidemiologia , Humanos , Medições Luminescentes/métodos , Fatores de Tempo , Uganda/epidemiologia , Proteínas Virais/imunologia
16.
Artif Intell Med ; 52(3): 153-63, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21571512

RESUMO

OBJECTIVES: Despite medical advances, infectious diseases are still a major cause of mortality and morbidity, disability and socio-economic upheaval worldwide. Early diagnosis, appropriate choice and immediate initiation of antibiotic therapy can greatly affect the outcome of any kind of infection. Phagocytes play a central role in the innate immune response of the organism to infection. They comprise the first-line of defense against infectious intruders in our body, being able to produce large quantities of reactive oxygen species, which can be detected by means of chemiluminescence (CL). The data preparation approach implemented in this work corresponds to a dynamic assessment of phagocytic respiratory burst localization in a luminol-enhanced whole blood CL system. We have previously applied this approach to the problem of identifying various intra-abdominal pathological processes afflicting peritoneal dialysis patients in the Nephrology department and demonstrated 84.6% predictive accuracy with the C4.5 decision-tree algorithm. In this study, we apply the CL-based approach to a larger sample of patients from two departments (Nephrology and Internal Medicine) with the aim of finding the most effective and interpretable feature sets and classification models for a fast and accurate identification of several infectious diseases. MATERIALS AND METHODS: Whole blood samples were collected from 78 patients (comprising 115 instances) with respiratory infections, infections associated with renal replacement therapy and patients without infections. CL kinetic parameters were calculated for each case, which was assigned into a specific clinical group according to the available clinical diagnostics. Feature selection wrapper and filter methods were applied to remove the irrelevant and redundant features and to improve the predictive performance of disease classification algorithms. Three data mining algorithms, C4.5 (J48) decision tree, support vector machines and naive Bayes classifier were applied for inducing disease classification models and their performance in classifying three clinical groups was evaluated by 10 runs of a stratified 10-fold cross-validation. RESULTS AND CONCLUSIONS: The results demonstrate that the predictive power of the best models obtained with the three evaluated algorithms after feature selection was found to be in the range of 63.38 ± 2.18-70.68 ± 1.43%. The highest disease classification accuracy was reached by C4.5, which also provides the most informative model in the form of a decision tree, and the lowest accuracy was obtained with naive Bayes. The feature selection method attaining the best classification performance was the wrapper method in forward direction. Moreover, the classification models exposed biological patterns specific to the clinical states and the predictive features selected were found to be characteristic of a specific disorder. Based on these encouraging results, we believe that the CL-based data pre-processing approach combined with the wrapper forward feature selection procedure and the C4.5 decision-tree algorithm has a clear potential to become a fast, informative, and sensitive tool for predictive diagnostics of infectious diseases in clinics.


Assuntos
Doenças Transmissíveis/classificação , Fagócitos/imunologia , Fagocitose , Idoso , Doenças Transmissíveis/sangue , Feminino , Humanos , Luminescência , Masculino , Pessoa de Meia-Idade
17.
Anal Chem ; 83(11): 4258-65, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21517122

RESUMO

Oftentimes the etiological diagnostic differentiation between viral and bacterial infections is problematic, while clinical management decisions need to be made promptly upon admission. Thus, alternative rapid and sensitive diagnostic approaches need to be developed. Polymorphonuclear leukocytes (PMNs) or phagocytes act as major players in the defense response of the host during an episode of infection, and thereby undergo functional changes that differ according to the infections. PMNs functional activity can be characterized by quantification and localization of respiratory burst production and assessed by chemiluminescent (CL) byproduct reaction. We have assessed the functional states of PMNs of patients with acute infections in a luminol-amplified whole blood system using the component CL approach. In this study, blood was drawn from 69 patients with fever (>38 °C), and diagnosed as mainly viral or bacterial infections in origin. Data mining algorithms (C4.5, Support Vector Machines (SVM) and Naïve Bayes) were used to induce classification models to distinguish between clinical groups. The model with the best predictive accuracy was induced using C4.5 algorithm, resulting in 94.7% accuracy on the training set and 88.9% accuracy on the testing set. The method demonstrated a high predictive diagnostic value and may assist the clinician one day in the distinction between viral and bacterial infections and the choice of proper medication.


Assuntos
Infecções Bacterianas/diagnóstico , Medições Luminescentes/métodos , Fagócitos/imunologia , Infecções por Vírus de RNA/diagnóstico , Doença Aguda , Algoritmos , Células Sanguíneas/imunologia , Humanos , Cinética , Luminol/química , Modelos Teóricos , Espécies Reativas de Oxigênio/metabolismo , Software
18.
BMC Cancer ; 8: 247, 2008 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-18721484

RESUMO

BACKGROUND: We have been studying the native humoral immune response to cancer and have isolated a library of fully human autoantibodies to a variety of malignancies. We previously described the isolation and characterization of two fully human monoclonal antibodies, 27.F7 and 27.B1, from breast cancer patients that target the protein known as GIPC1, an accessory PDZ-domain binding protein involved in regulation of G-protein signaling. Human monoclonal antibodies, 27.F7 and 27.B1, to GIPC1 demonstrate specific binding to malignant breast cancer tissue with no reactivity with normal breast tissue. METHODS: The current study employs cELISA, flow cytometry, Western blot analysis as well as immunocytochemistry, and immunohistochemistry. Data is analyzed statistically with the Fisher one-tail and two-tail tests for two independent samples. RESULTS: By screening several other cancer cell lines with 27.F7 and 27.B1 we found consistently strong staining of other human cancer cell lines including SKOV-3 (an ovarian cancer cell line). To further clarify the association of GIPC1 with breast and ovarian cancer we carefully studied 27.F7 and 27.B1 using immunocytochemical and immunohistochemical techniques. An immunohistochemical study of normal ovarian tissue, benign, borderline and malignant ovarian serous tumors, and different types of breast cancer revealed high expression of GIPC1 protein in neoplastic cells. Interestingly, antibodies 27.F7 and 27.B1 demonstrate differential staining of borderline ovarian tumors. Examination of different types of breast cancer demonstrates that the level of GIPC1 expression depends on tumor invasiveness and displays a higher expression than in benign tumors. CONCLUSION: The present pilot study demonstrates that the GIPC1 protein is overexpressed in ovarian and breast cancer, which may provide an important diagnostic and prognostic marker and will constitute the basis for further study of the role that this protein plays in malignant diseases. In addition, this study suggests that human monoclonal antibodies 27.F7 and 27.B1 should be further evaluated as potential diagnostic tools.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Autoanticorpos/química , Neoplasias da Mama/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Anticorpos Monoclonais/química , Mama/metabolismo , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imuno-Histoquímica/métodos , Neoplasias Ovarianas/imunologia , Projetos Piloto
19.
BMC Cancer ; 8: 248, 2008 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-18721486

RESUMO

BACKGROUND: We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients. METHODS: The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification. RESULTS: By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein. CONCLUSION: We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/química , Anticorpos Monoclonais/química , Antígenos de Neoplasias/química , Autoanticorpos/química , Regulação Neoplásica da Expressão Gênica , Autoimunidade , Neoplasias da Mama , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , Imuno-Histoquímica/métodos , Cinética , Estrutura Terciária de Proteína
20.
Anal Chem ; 80(13): 5131-8, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18510343

RESUMO

Recurrent bacterial peritonitis is a major complication in peritoneal dialysis (PD) patients, which is associated with polymorphonuclear leukocyte (PMN) functional changes and can be assessed by a chemiluminescent (CL) reaction. We applied a new approach of a dynamic component chemiluminescence sensor for the assessment of functional states of PMNs in a luminol-amplified whole-blood system. This method is based on the evaluation of CL kinetic patterns of stimulated PMNs, while the parallel measurements of intracellular and extracellular production of reactive oxygen species (ROS) from the same sample can be conducted. Blood was drawn from diabetic and nondiabetic patients during follow-up, and during peritonitis. Healthy medical personnel served as the control group. Chemiluminescence curves were recorded and presented as a sum of three biological components. CL kinetic parameters were calculated, and functional states of PMNs were assessed. Data mining algorithms were used to build decision tree models that can distinguish between different clinical groups. The induced classification models were used afterward for differentiating and classifying new blind cases and demonstrated good correlation with medical diagnosis (84.6% predictive accuracy). In conclusion, this novel method shows a high predictive diagnostic value and may assist in detection of PD-associated clinical states.


Assuntos
Medições Luminescentes/métodos , Neutrófilos/fisiologia , Diálise Peritoneal/métodos , Peritonite/sangue , Diabetes Mellitus/sangue , Feminino , Humanos , Medições Luminescentes/instrumentação , Luminol/química , Masculino , Pessoa de Meia-Idade , Diálise Peritoneal/efeitos adversos , Peritonite/diagnóstico , Projetos Piloto , Espécies Reativas de Oxigênio/sangue , Explosão Respiratória
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