| 2021 | Transgenic plants expressing a Clostridium difficile spore antigen as an approach to develop low-cost oral vaccines. | Biotechnol Prog | Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low-grade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant-based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB-CdeM), carrying the CdeM antigen, fused to the carboxi-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB-CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB-CdeM antigen in the range of 52-90 μg/g dry weight leaf tissues. The antigenicity of the plant-made LTB-CdeM antigen was evidenced by GM1-ELISA and immunogenicity assessment performed in test mice revealed that the LTB-CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant-based vaccines against C. difficile infection. |
| 2019 | Mechanisms of African swine fever virus pathogenesis and immune evasion inferred from gene expression changes in infected swine macrophages. | PLoS One | African swine fever (ASF) is a swine disease caused by a large, structurally complex, double-stranded DNA virus, African swine fever virus (ASFV). In domestic pigs, acute infection by highly virulent ASF viruses causes hemorrhagic fever and death. Previous work has suggested that ASFV pathogenesis is primarily mediated by host cytokines produced by infected monocytes and macrophages. To better understand molecular mechanisms mediating virus pathogenesis and immune evasion, we used transcriptome analysis to identify gene expression changes after ASFV infection in ex vivo swine macrophages. Our results suggest that the cytokines of TNF family including FASLG, LTA, LTB, TNF, TNFSF4, TNFSF10, TNFSF13B and TNFSF18 are the major causative cytokine factors in ASF pathogenesis via inducing apoptosis. Other up-regulated proinflammatory cytokines (IL17F and interferons) and down-regulated anti-inflammatory cytokine (IL10) may also significantly contribute to ASF pathogenesis and cause excessive tissue inflammatory responses. The differential expression of genes also indicates that ASFV could evade both the innate and adaptive immune responses by (i) inhibiting MHC Class II antigen processing and presentation, (ii) avoiding CD8+ T effector cells and neutrophil extracellular traps via decreasing expression of neutrophil/CD8+ T effector cell-recruiting chemokines, (iii) suppressing M1 activation of macrophages, (iv) inducing immune suppressive cytokines, and (v) inhibiting the processes of macrophage autophagy and apoptosis. These results provide novel information to further investigate and better understand the mechanism of pathogenesis and immune evasion of this devastating swine disease. |
| 2019 | Assessment of disease specific immune responses in enteric diseases using dried blood spot (DBS). | PLoS One | Blood collection, transportation and storage remain a problem in countries where infrastructure, laboratory facilities and skilled manpower are scarce. This limits evaluation of immune responses in natural infections and vaccination in field studies. We developed methods to measure antigen specific antibody responses using dried blood spot (DBS) in cholera, ETEC and typhoid fever patients as well as recipients of oral cholera vaccine (OCV).We processed heparinized blood for preparing DBS and plasma specimens from patients with, cholera, ETEC and typhoid as well as OCV recipients. We optimized the conventional vibriocidal method to measure vibriocidal antibody response in DBS eluates. We measured responses in DBS samples and plasma (range of titer of 5 to 10240). Vibriocidal titer showed strong agreement between DBS eluates and plasma in cholera patients (ICC = 0.9) and in OCV recipients (ICC = 0.8) using the Bland-Altman analysis and a positive correlation was seen (r = 0.7, p = 0.02 and r = 0.6, p = 0.006, respectively). We observed a strong agreement of lipopolysaccharide (LPS) and cholera toxin B (CTB)-specific antibody responses between DBS eluates and plasma in cholera patients and OCV recipients. We also found agreement of heat labile toxin B (LTB) and membrane protein (MP)-specific antibody responses in DBS eluates and plasma specimen of ETEC and typhoid patients respectively.Our results demonstrate that dried blood specimens can be used as an alternate method for preservation of samples to measure antibody responses in enteric diseases and vaccine trials and can be applied to assessment of responses in humanitarian crisis and other adverse field settings. |
| 2019 | The utility of real-time polymerase chain reaction in detecting Coccidioides immitis among clinical specimens in the Central California San Joaquin Valley. | Med Mycol | Coccidioidomycosis, the fungal infection caused by dimorphic Coccidioides species, is typically diagnosed by histopathologic identification of spherules, by culture, or by serology. These tests are reliable but time-intensive, delaying diagnosis and treatment. Rapid real-time polymerase chain reaction (RT-PCR) can be performed and was validated to identify Coccidioides immitis using an in-house developed assay for the Becton Dickinson molecular instrument (BD MAXTM). These studies were performed using patient samples that had been shown to be positive on previously set up fungal cultures. To evaluate this new RT-PCR test in the clinical setting, we conducted a retrospective chart review of patients (N = 1160) who underwent Coccidioides PCR (Cocci PCR) on clinical samples between March 1, 2014, and Dec 31, 2016. We abstracted clinical, microbiologic, serologic, radiographic, treatment, and follow-up data. Specimens of cerebrospinal fluid (CSF), bronchioalveolar lavage fluid (BAL), lung tissue biopsy (LTB), sputum, and pleural fluid were evaluated to determine sensitivity and specificity. Of the 113 specimens that tested positive for Cocci PCR, all had clinical disease defined by traditional clinical criteria, yielding 100% specificity. Overall sensitivity was 74% versus 46% for fungal culture and was available in 4 hours rather than 1-2 weeks. Sensitivities varied by source material and clinical setting. CSF had a sensitivity of 59%, BAL for acute pneumonia 91%, sputum for acute pneumonia 94%, pleural fluid 86%, but LTB for lung nodules only 44%. Overall positive predictive value (PPV) was 100%, while negative predictive value (NPV) was 96%, but again this varied by specimen and clinical setting. Our experience with clinical testing of >1160 specimens over 2-3 years shows we can utilize this technology to improve our ability to diagnose disease but that the sensitivity varies by specimen source and clinical setting. |
| 2017 | Construction of an inactivated typhoid vaccine candidate expressing heat-labile enterotoxin B subunit and evaluation of its immunogenicity in a murine model. | J Med Microbiol | Typhoid fever caused by serovar Typhi has contributed to the global public health burden, particularly in developing countries. In this study, an . Typhi ghost was developed and its capacity as a vaccine candidate against typhoid fever was assessed. An plasmid pJHL187 harbouring a ghost cassette comprising the PhiX 174 lysis gene tightly controlled under the convergent promotor system was transformed into an gene-deleted mutant Typhi strain (STG). The gene encoding the heat-labile enterotoxin (LTB) protein was subcloned into a foreign antigen delivery cassette of pJHL187 to increase mucosal immunity. The stringent repression and expression of the lethal lysis gene in the system allowed stable production of the ghost strain and secretion of LTB, which was confirmed by immune blot analysis. The level of IgG and sIgA was significantly increased in the mice subcutaneously immunized with STG-LTB compared to the non-immunized mice (<0.05). The CD3CD4 T cell subpopulation was augmented in the immunized group (<0.05) and showed the increment of immunomodulatory cytokines IL-2, IL-6, IL-12, IL-17 and IFN-γ in restimulated splenocytes isolated from the inoculated mice. The serum bactericidal activity of antibodies generated in the rabbits injected with STG-LTB was proved by the elimination of approximately 87.5 % of wild-type . Typhi in the presence of exogenous complement. The results demonstrated that the STG-LTB ghost effectively enhanced the immunological responses, meaning that STG-LTB is potentially available as a vaccine candidate against typhoid fever. |
| 2013 | Tuberculosis in anti-TNF-α treated patients remains a problem in countries with an intermediate incidence: analysis of 25 patients matched with a control population. | J Crohns Colitis | An increased incidence of tuberculosis (TB) in patients under anti-TNF-α therapy has been reported, but outcome compared with TB in the general population are unknown.Patients who had active tuberculosis while taking anti-TNF-α drugs were studied and compared with a control group of community-acquired TB matched for sex, age and data of TB.Twenty-five cases of TB were reported from a cohort of 765 patients under anti-TNF-α from 2001 to 2012. The incidence of TB per 100,000 patient-years was estimated to be 1337, 792 and 405 respectively for those on infliximab, adalimumab and etanercept. Twelve patients had inflammatory bowel disease, ten had rheumatologic diseases and three had psoriasis. From the 17 patients screened for latent TB before anti-TNF-α, three were treated with isoniazid. TB was diagnosed 1-108 months after starting anti-TNF-α, being the median time six, seven and 89 months respectively for those on infliximab, adalimumab and etanercept. Sixty per cent of the cases had extra-pulmonary TB. No deaths occurred in the case groups, while two died in control TB patients. Patients on anti-TNF-α drugs had more frequent extra-pulmonary TB, fever on presentation, higher mean C-reactive protein and lower positive rate of acid-fast bacilli.TB may still occur in those with negative testing, some of them probably representing new infections instead of reactivations. Three out of 25 patients had TB in spite of previously treated LTB, although, the outcome of TB was not worse than in the general population. |
| 2011 | In vivo and in vitro pharmacological activity of Aristolochia tagala (syn: Aristolochia acuminata) root extracts. | Pharm Biol | Aristolochia tagala Cham. (syn: Aristolochia acuminata Lam.) (Aristolochiaceae), known as Nallayishwari in Telugu, has been of interest to researchers because of its traditional uses for treating rheumatic pains and fever.The anti-inflammatory activity of the petroleum ether, ethyl acetate, and ethanol extracts of A. tagala roots were investigated for the first time.In vivo and in vitro anti-inflammatory effects were investigated employing the carrageenan-induced hind paw edema in rats and the macrophage cell line RAW264.7 stimulated with proinflammatory stimuli (lipopolysaccharide interferon γ or the calcium ionophore A23187) to determine PGE(2) or LTB(4) release, respectively.All the extracts exhibited anti-inflammatory effects which were found to be significant (p < 0.001) at 200 and 400 mg/kg, p.o, in rats tested and the ethyl acetate extract inhibited the induction of PGE(2) with IC(50) = 39.1 mg mL(-1) and LTB(4) with IC(50) = 29.5 mg mL(-1).These findings demonstrate that the A. tagala roots have excellent anti-inflammatory activity and validate the traditional indications of this plant in its origin country. |
| Efficacy of the scoring system, recommended by the Brazilian National Ministry of Health, for the diagnosis of pulmonary tuberculosis in children and adolescents, regardless of their HIV status. | J Bras Pneumol | To determine the efficacy of the scoring system, recommended by the Brazilian National Ministry of Health (NMH), for the diagnosis of pulmonary tuberculosis (TB) in children and adolescents, regardless of their HIV status.This was a cross-sectional analytical study carried out between January of 2002 and December of 2006, involving 239 individuals less than 15 years of age. The patients were divided into four groups: latent TB (LTB group; n = 81); no-TB (NTB group; n = 41); TB group (n = 104); and TB/HIV group (n = 13). We studied the clinical, radiological and laboratory findings according to the scoring system.Reports of fever, cough, asthenia and weight loss for at least two weeks were significantly higher in the TB group (p < 0.0001). The proportion of cases with a history of any contact and household contact with a TB patient was, respectively, 95.0% and 86.1% in the TB group, versus 75.0% and 58.3% in the TB/HIV group. In the TB and TB/HIV groups, respectively, chest X-rays revealed parenchymal alterations in 75.0% and 53.9%, revealing combined parenchymal/lymph node alterations in 18.2% and 30.8%. There were no significant differences among the groups regarding the tuberculin skin test results. In the TB group, 16.3% of the patients were malnourished (p < 0.005 vs. the LTB group). The mean NMH system scores in the LTB, NTB, TB and TB/HIV groups were, respectively, 24.2, 18.5, 45.3 and 41.5.The NMH system scores were significantly higher in the TB and TB/HIV groups than in the other two groups. Therefore, this scoring system was valid for the diagnosis of pulmonary TB in this population, regardless of HIV status. |
| An experimentally induced Chlamydia suis infection in pigs results in severe lung function disorders and pulmonary inflammation. | Vet Res | This study was aimed at evaluating the pathophysiology of pulmonary dysfunctions and inflammatory consequences of an acute respiratory chlamydial infection induced experimentally in conventionally raised pigs (aged 39-44 days). Eight animals were exposed to Chlamydia suis (C. suis) and four non-infected animals served as controls. The total observation period was from seven days before challenge to seven days post exposure. While non-infected control pigs did not exhibit any clinical symptoms, animals exposed to C. suis developed fever and were severely respiratory distressed within the first week after exposure. After C. suis infection, pulmonary dysfunctions were characterised by a significant decrease in the diffusion capacity of the lung (i.e. transfer factor of the lung for carbon monoxide; TL CO), a significant increase in the functional residual capacity (FRC), and significant changes in the pattern of ventilation (respiratory rate increased while the tidal volume decreased). In exhaled breath condensate (EBC), leukotriene B(4) (LTB(4)) and interleukin 6 (IL-6) showed a tendency to increase after infection. In the broncho-alveolar lavage fluid (BALF) of C. suis infected pigs, the activity of matrix metalloprotease 9 (MMP-9) was found to be increased compared to controls. BALF cytology was characterised by increased numbers of granulocytes and activated lymphocytes. Pulmonary inflammation in infected pigs was confirmed by post mortem histology. A prominent dissemination of chlamydial bodies in the lung was accompanied by an influx of macrophages, granulocytes and activated T-cells. Data obtained in this study provide new insight into the pathogenesis of acute respiratory chlamydial infections in pigs. |