Bactericidal activity of M protein conserved region antibodies against group A streptococcal isolates from the Northern Thai population

BACKGROUND: Most group A streptococcal (GAS) vaccine strategies have focused on the surface M protein, a major virulence factor of GAS. The amino-terminus of the M protein elicits antibodies, that are both opsonic and protective, but which are type specific. J14, a chimeric peptide that contains 14 amino acids from the M protein conserved C-region at the carboxy-terminus, offers the possibility of a vaccine which will elicit protective opsonic antibodies against multiple different GAS strains. In this study, we searched for J14 and J14-like sequences and the number of their repeats in the C-region of the M protein from GAS strains isolated from the Northern Thai population. Then, we examined the bactericidal activity of J14, J14.1, J14-R1 and J14-R2 antisera against multiple Thai GAS strains. RESULTS: The emm genes of GAS isolates were sequenced and grouped as 14 different J14-types. The most diversity of J14-types was found in the C1-repeat. The J14.1 type was the major sequence in the C2 and C3-repeats. We have shown that antisera raised against the M protein conserved C-repeat region peptides, J14, J14.1, J14-R1 and J14-R2, commonly found in GAS isolates from the Northern Thai population, are able to kill GAS of multiple different emm types derived from an endemic area. The mean percent of bactericidal activities for all J14 and J14-like peptide antisera against GAS isolates were more than 70%. The mean percent of bactericidal activity was highest for J14 antisera followed by J14-R2, J14.1 and J14-R1 antisera. CONCLUSION: Our study demonstrated that antisera raised against the M protein conserved C-repeat region are able to kill multiple different strains of GAS isolated from the Northern Thai population. Therefore, the four conserved "J14" peptides have the potential to be used as GAS vaccine candidates to prevent streptococcal infections in an endemic area

M protein typing of Thai group A streptococcal isolates by PCR-Restriction fragment length polymorphism analysis

BACKGROUND: Group A streptococcal (GAS) infections can lead to the development of severe post-infectious sequelae, such as rheumatic fever (RF) and rheumatic heart disease (RHD). RF and RHD are a major health concern in developing countries, and in indigenous populations of developed nations. The majority of GAS isolates are M protein-nontypeable (MNT) by standard serotyping. However, GAS typing is a necessary tool in the epidemiologically analysis of GAS and provides useful information for vaccine development. Although DNA sequencing is the most conclusive method for M protein typing, this is not a feasible approach especially in developing countries. To overcome this problem, we have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based assay for molecular typing the M protein gene (emm) of GAS. RESULTS: Using one pair of primers, 13 known GAS M types showed one to four bands of PCR products and after digestion with Alu I, they gave different RFLP patterns. Of 106 GAS isolates examined from the normal Thai population and from patients with GAS-associated complications including RHD, 95 isolates gave RFLP patterns that corresponded to the 13 known M types. Only 11 isolates gave RFLP patterns that differed from the 13 known M types. These were then analyzed by DNA sequencing and six additional M types were identified. In addition, we found that M93 GAS was the most common M type in the population studied, and is consistent with a previous study of Thai GAS isolates. CONCLUSION: PCR-RFLP analysis has the potential for the rapid screening of different GAS M types and is therefore considerably advantageous as an alternative M typing approach in developing countries in which GAS is endemic

Staying Ahead of the Digital Technological Curve Using Survey Methods

In the early twentieth century, surveys were an innovative and neoteric methodology. Collected in-person or by mail, researchers could ascertain the thoughts and opinions of a small sample, which could then be applied to the general population. Almost one hundred years later, the use of surveys has become pervasive in society due to digital technological advancement. However, while the digital evolution has not only altered the possibilities of how, when, and where surveys may be administered, the threats to this methodology has also evolved. While issues related to previously known errors (i.e. sampling error, non-response error, etc.) remain and have also evolved, new threats regarding confidentiality and privacy, design issues, and others, have emerged in response to this digital advancement. Novice and experienced researchers alike should be cognizant of the impact digital technologies have had on survey data collection to ensure high quality research findings. This paper explores the threats to survey methodology due to digital technological changes and discusses how novice researchers and students can mitigate these challenges. Au début du vingtième siècle, les sondages constituaient une méthodologie novatrice et moderne. Les chercheurs pouvaient, à partir d’un petit échantillon, cerner des idées et opinions recueillies en personne ou par courrier, pour ensuite les appliquer à la population générale. Près de 100 ans plus tard, grâce aux avancées de la technologie numérique, les sondages sont omniprésents dans la société. Or, l’évolution numérique a non seulement modifié les paramètres spatiotemporels et les techniques d’administration des sondages, mais aussi les risques liés à cette méthodologie. Alors que les problèmes liés aux erreurs déjà connues (erreurs d’échantillonnage, erreurs de non-réponse, etc.) demeurent et évoluent tout autant, de nouvelles menaces associées à la confidentialité et à la protection des renseignements personnels, aux questions de conception et à d’autres enjeux ont surgi en réaction à cette évolution technologique. Afin d’optimiser la qualité des résultats de recherche, les chercheuses débutantes et chevronnées devraient être au fait des retombées possibles des technologies numériques sur la collecte de données par sondage. Cet article porte sur les menaces à la méthodologie des sondages en raison des changements liés aux technologies numériques, et traite de stratégies permettant aux étudiantes et aux chercheuses débutantes d’éviter ces écueils

Apoptosis and expression of cytotoxic T lymphocyte effector molecules in renal allografts

Cytotoxic T lymphocyte (CTL) mediated apoptosis is thought to play a major role in the rejection of renal allografts following transplantation, however, the CTL effector mechanism that is primarily responsible for immunological rejection is unknown. The two major effector pathways of CTL killing which lead to apoptosis involve the Fas/Fas ligand (Fas L) lytic pathway, and the perforin/granzyme degranulation pathway. The expression of Cn effector molecules which influence these pathways include Pas, Pas L and TiA-1 (cytotoxic granule protein). This study has investigated apoptosis by in situ terminal deoxytransferase-catalysed DNA nick end labelling (TUNEL), and the expression of CTL effector molecules by immunohistochemistry, in renal allograft biopsies obtained from patients following kidney transplantation. Renal biopsies were classified into three histological groups; acute cellular rejection, chronic rejection, or no rejection. The extent of T-cell infiltration of renal tissues was assessed by immunohistochemical staining with an anti-CD3 monoclonal antibody. Numerous TUNEL positive cells were detected in all transplant biopsies examined; these consisted mainly of renal tubular cells and infiltrating cells, with some TUNEL positive cells also detected in the glomeruli. In the case of normal kidney tissue, renal cells also stained positive for TUNEL but there was no lymphocytic infiltration. There was significantly more T-cell infiltration observed in acute rejection biopsies compared to the no rejection biopsies. In the case of Pas L expression, there was little expression in all three biopsy groups, apart from one case of chronic rejection. Conversely, although there were no significant differences in TiA-1 expression between the three biopsy groups, TiA-1 expression was more prominent in acute rejection biopsies. Furthermore, Pas expression was significantly decreased in acute rejection biopsies when compared to those of chronic and no rejection in which Fas was predominantly localized in the renal tubular cells. These results indicate that the mechanism of CTL killing leading to the rejection of renal allografts may be different in acute and chronic rejection. Moreover, our data indicate the potential for cytotoxic granule-based CTL killing in acute renal allograft rejection but not in chronic rejection

B- and T-Cell Responses in Group A Streptococcus M-Protein- or Peptide-Induced Experimental Carditis▿

The etiology of rheumatic fever and rheumatic heart disease (RF/RHD) is believed to be autoimmune, involving immune responses initiated between streptococcal and host tissue proteins through a molecular mimicry mechanism(s). We sought to investigate the humoral and cellular responses elicited in a Lewis rat model of group A streptococcus M-protein- or peptide-induced experimental valvulitis/carditis, a recently developed animal model which may, in part, represent human rheumatic carditis. Recombinant streptococcal M5 protein elicited opsonic antibodies in Lewis rats, and anti-M5 antisera recognized epitopes within the B- and C-repeat regions of M5. One peptide from the streptococcal M5 protein B-repeat region (M5-B.6, amino acids 161 to 180) induced lymphocytes that responded to both recombinant M5 and cardiac myosin. Rats immunized with streptococcal M5 protein developed valvular lesions, distinguished by infiltration of CD3+, CD4+, and CD68+ cells into valve tissue, consistent with human studies that suggest that RF/RHD are mediated by inflammatory CD4+ T cells and CD68+ macrophages. The current study provides additional information that supports the use of the rat autoimmune valvulitis model for investigating RF/RHD

Oral vaccine delivery - New strategies and technologies

Although most commercial vaccines are delivered by injection, there is an increasing interest in needle-free vaccine delivery for reasons including the ability to elicit immune responses at mucosal surfaces, ease of administration, and the ability to administer vaccines without the need for trained medical professionals. This review summarizes strategies and technologies that are being used to improve oral vaccine absorption. Peptides and proteins, which comprise important vaccine components, exhibit unfavorable physicochemical properties including degradation in the gastrointestinal tract, and poor transport across the intestinal wall, which hinder oral vaccine development. Approaches to overcome these obstacles aim to provide new vaccines and delivery systems that are capable of eliciting protective immune responses, and are making an impact on current vaccine development

Expression of cytokine mRNA transcripts in renal cell carcinoma

Renal cell carcinoma (RCC) is a solid tumour of the kidney and is the most common renal neoplasm. Despite the presence of tumour infiltrating lymphocytes (TIL) in RCC, these tumours continue to progress in vivo suggesting a poor host immune response to the tumour, and the suppression of TIL effector function. Cytokines are key molecules that modulate the function of T cells. The possibility is investigated that the local production of cytokines in RCC contributes to immunosuppression of TIL. The expression of pro-inflammatory (IFN-γ/IL-2) and immunosuppressive (IL-10/TGF-β) cytokine mRNA transcripts was determined in RCC, normal kidney and peripheral blood of RCC patients using a semi-quantitative reverse transcriptase-polymerase chain reaction (RTPCR) with cytokine-specific primers. Following Southern blot hybridization of the PCR products with internal radiolabelled oligonucleotide probes, cytokine transcript levels were measured by densitometry and expressed relative to the glyceraldehyde-3-phosphate dehydrogenase densitometry score. With the exception of IL-10, there were no differences in expression of cytokine mRNA transcripts between the peripheral blood of patients and normal healthy individuals. It was found that TGF-β transcripts were well represented in normal kidney and RCC. In contrast, the expression of IFN-γ transcripts, while low in the majority of samples, was significantly increased in RCC when compared to normal kidney (P = 0.05). The IL-2 and IL-10 transcripts showed a more variable expression in normal kidney and RCC, with no significant differences in expression between the sample groups. The data demonstrating pro-inflammatory and immunosuppressive cytokine expression in RCC do not support a prominent immunosuppressive cytokine profile in these tumours