Photobiomodulation inhibits inflammation in the temporomandibular joint of rats.

Photobiomodulation (PBM) has been applied as a non-invasive technique for treating temporomandibular joint symptoms, especially on painful condition's relief, however the anti-inflammatory mechanism underlying the effect of PBM remains uncertain. This study aims to evaluate the mechanisms of action of PBM (808 nm) in a carrageenan-induced inflammation on temporomandibular joint (TMJ) of rats. In this study male Wistar rats were pre-treated with irradiation of a low-power diode laser for 15 s on TMJ (infra-red 808 nm, 100 mW, 50 J/cm2 and 1.5 J) 15 min prior an injection in the temporomandibular joint of carrageenan (100 µg/TMJ). 1 h after the TMJ treatments, the rats were terminally anesthetized for joint cavity wash and periarticular tissues collect. Samples analysis demonstrated that PBM inhibit leukocytes chemotaxis in the TMJ and significantly reduces amounts of TNF-α, IL-1ß and CINC-1. In addition, Western blotting analysis demonstrated that PBM significantly decreased the protein levels of P2X3 and P2X7 receptors in the periarticular tissues. On the other hand, PBM was able to increase protein level of IL-10 (anti-inflammatory cytokine). In summary, it is possible to suggest that PBM inhibit inflammatory chemotaxis, modulation the balance of the pro- and anti-inflammatory characteristics of inflammatory cells.

Biofilms busters to improve the detection of Borrelia using PCR.

Lyme disease is an affection caused by a spirochete infection called Borrelia Burgdorferi which may harbor a varied and misleading clinical symptomatology. The serology tests commonly used for diagnosis show a wide sensitivity varying from 34% to 70,5%, leaving many infected patients with false negative tests. Alternative techniques such as polymerase chain reaction (PCR) could be helpful but not conclusive enough. Using biofilm busters, such as stevia and serratiopeptidase, could lead to bacterial blood release, thus increasing the spirochete load, making PCR test more sensitive, thus improving the patient's diagnosis and management.

Enzyme-Linked Immunospot Assay as a Complementary Method to Assess and Monitor Cytomegalovirus Infection in Kidney Transplant Recipients on Pre-emptive Antiviral Therapy: A Single-Center Experience.

BACKGROUND: Cytomegalovirus (CMV) disease represents a major cause of post-transplantation morbidity and mortality. To estimate the risk of infection and monitor response to antiviral therapy, current guidelines suggest combination of viral load monitoring with direct assessment of CMV-specific immune response. We used enzyme-linked immunospot (ELISpot) for the evaluation of CMV-specific T-cell response in kidney transplant recipients with CMV viremia and investigated how information gained could help manage CMV infection. METHODS: Seventeen patients on pre-emptive antiviral therapy and CMV quantitative polymerase chain reaction (qPCR) ≥500 copies/mL (first episode after transplantation) were assessed using ELISpot and divided into Weak (9 patients with baseline ELISpot <25 spot-forming colonies [SFCs]/200,000 peripheral blood mononuclear cells [PBMCs]) and Strong Responders (8 patients with baseline ELISpot ≥25 SFCs/200,000 PBMCs). CMV-specific T-cell response, infection severity, viral load, and antiviral therapy were prospectively recorded and compared between groups at 1, 2, and 24 months of follow-up. RESULTS: Demographic and transplant characteristics of Weak and Strong Responders were similar. No episodes of CMV disease were observed. Weak Responders were more likely to experience CMV syndrome (56% vs 36.5%) and late virus reactivation (56% vs 25%) than Strong Responders. Weak Responders showed higher baseline median viral loads (19,700 vs 9265 copies/mL) and needed antiviral therapy for longer (179 vs 59.5 days). T-cell response showed 2 main patterns: early and delayed. CONCLUSIONS: ELISpot provides prognostic information about infection severity, risk of late reactivation, and response to therapy. Randomized trials, evaluating the need for antiviral therapy in kidney transplant recipients with asymptomatic infection and effective virus-specific T-cell immune response, are warranted.

Differential effects of FTY720 on the B cell compartment in a mouse model of multiple sclerosis.

BACKGROUND: MP4-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), which enables targeted research on B cells, currently much discussed protagonists in MS pathogenesis. Here, we used this model to study the impact of the S1P1 receptor modulator FTY720 (fingolimod) on the autoreactive B cell and antibody response both in the periphery and the central nervous system (CNS). METHODS: MP4-immunized mice were treated orally with FTY720 for 30 days at the peak of disease or 50 days after EAE onset. The subsequent disease course was monitored and the MP4-specific B cell/antibody response was measured by ELISPOT and ELISA. RNA sequencing was performed to determine any effects on B cell-relevant gene expression. S1P1 receptor expression by peripheral T and B cells, B cell subset distribution in the spleen and B cell infiltration into the CNS were studied by flow cytometry. The formation of B cell aggregates and of tertiary lymphoid organs (TLOs) was evaluated by histology and immunohistochemistry. Potential direct effects of FTY720 on B cell aggregation were studied in vitro. RESULTS: FTY720 significantly attenuated clinical EAE when treatment was initiated at the peak of EAE. While there was a significant reduction in the number of T cells in the blood after FTY720 treatment, B cells were only slightly diminished. Yet, there was evidence for the modulation of B cell receptor-mediated signaling upon FTY720 treatment. In addition, we detected a significant increase in the percentage of B220+ B cells in the spleen both in acute and chronic EAE. Whereas acute treatment completely abrogated B cell aggregate formation in the CNS, the numbers of infiltrating B cells and plasma cells were comparable between vehicle- and FTY720-treated mice. In addition, there was no effect on already developed aggregates in chronic EAE. In vitro B cell aggregation assays suggested the absence of a direct effect of FTY720 on B cell aggregation. However, FTY720 impacted the evolution of B cell aggregates into TLOs. CONCLUSIONS: The data suggest differential effects of FTY720 on the B cell compartment in MP4-induced EAE.

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy.

Ex vivo functional immunoassays such as ELISpot and intracellular cytokine staining (ICS) by flow cytometry are crucial tools in vaccine development both in the identification of novel immunogenic targets and in the immunological assessment of samples from clinical trials. Cryopreservation and subsequent thawing of PBMCs via validated processes has become a mainstay of clinical trials due to processing restrictions inherent in the disparate location and capacity of trial centres, and also in the need to standardize biological assays at central testing facilities. Logistical and financial requirement to batch process samples from multiple study timepoints are also key. We used ELISpot and ICS assays to assess antigen-specific immunogenicity in blood samples taken from subjects enrolled in a phase II malaria heterologous prime-boost vaccine trial and showed that the freeze thaw process can result in a 3-5-fold reduction of malaria antigen-specific IFNγ-producing CD3+CD4+ effector populations from PBMC samples taken post vaccination. We have also demonstrated that peptide responsive CD8+ T cells are relatively unaffected, as well as CD4+ T cell populations that do not produce IFNγ. These findings contribute to a growing body of data that could be consolidated and synthesised as guidelines for clinical trials with the aim of increasing the efficiency of vaccine development pipelines.

Positive selection of type II collagen-reactive CD80high marginal zone B cells in DBA/1 mice.

To investigate whether dysregulated selection of autoreactive marginal zone (MZ) B cells is involved in autoimmune diseases, we examined MZ B cell profile in multiple strains of mice, and found that type II collagen (CII)-reactive autoreactive CD80high MZ B cells spontaneously developed in the DBA/1, but not in C57BL/6 mice. CD80high MZ B cells that were characteristically found in DBA/1 mice expressed higher levels of TACI, SLAM3, and SLAM6 than the usual CD80low MZ B cells. Notably, the CD80high MZ B cells were more sensitive to ibrutinib, a Bruton's tyrosine kinase inhibitor, than CD80low MZ or follicular B cells and their transient depletion via intravenous injection of ibrutinib significantly delayed the induction of collagen-induced arthritis (CIA). In summary, we suggest that the positive selection of CII-reactive CD80high MZ B cells is a critical homeostatic process predisposing the DBA/1 mice to the CIA induction.

Low hemagglutinin antigen dose influenza vaccines adjuvanted with AS03 alter the long-term immune responses in BALB/c mice.

We investigated the long-term immune profiles of dose-sparing, AS03-adjuvanted vaccines compared to a traditional high-dose, unadjuvated influenza vaccine formulation. BALB/c mice received 2 IM injections of influenza A/Uruguay/716/2007 (H3N2) split vaccine antigen: high-dose (HD) (3 µg hemagglutinin (HA)/dose) or low-dose (LD) formulations (0.03 µg or 0.003 µg HA) with AS03 and were followed to 34 weeks post-boost (pb). We examined serologic responses, spleen and bone marrow (BM) HA-specific antibody-secreting cells (ASCs) by ELISpot, influenza-specific cytokine/chemokine production in re-stimulated splenocytes by multiplex ELISA, and antigen-specific CD4+ T cells that express cytokines (IL-2, IFNγ, TNFα and IL-5) by flow cytometry. All formulations elicited robust serum antibody titers that persisted for at least 34 weeks. The number of antigen-specific ASCs in the spleen and BM were higher in the 2 LD +AS03 groups, but despite having fewer ASCs, the average spot size in the HD-unadjuvanted group was larger at later time-points, suggesting greater antibody production per cell. Striking differences in the long-term profiles induced by the different vaccine formulations may contribute to these different ASC profiles. The HD-unadjuvanted vaccine elicited strong Th2 cytokines during the first 6 weeks pb but LD+AS03 groups generated broader, more durable responses at later timepoints. Finally, the 0.03 µg HA+AS03 group generated the greatest number of antigen-specific CD4+ T cells and the highest percentage of poly-functional cells that expressed 2 or more cytokines. Although all of the tested vaccines induced durable antibody responses, we show that different vaccine formulations (dose-sparing, adjuvant) generate distinct long-term immune profiles. Furthermore, our data suggest that the different profiles may be generated through unique mechanisms.

A novel multi-drug metronomic chemotherapy significantly delays tumor growth in mice.

BACKGROUND: The tumor immunosuppressive microenvironment represents a major obstacle to an effective tumor-specific cellular immune response. METHODS: In the present study, the counterbalance effect of a novel metronomic chemotherapy protocol on such an immunosuppressive microenvironment was evaluated in a mouse model upon sub-cutaneous ectopic implantation of B16 melanoma cells. The chemotherapy consisted of a novel multi-drug cocktail including taxanes and alkylating agents, administered in a daily metronomic fashion. The newly designed strategy was shown to be safe, well tolerated and significantly efficacious. RESULTS: Treated animals showed a remarkable delay in tumor growth and prolonged survival as compared to control group. Such an effect was directly correlated with CD4(+) T cell reduction and CD8(+) T cell increase. Furthermore, a significant reduction in the percentage of both CD25(+)FoxP3(+) and CD25(+)CD127(low) regulatory T cell population was found both in the spleens and in the tumor lesions. Finally, the metronomic chemotherapy induced an intrinsic CD8(+) T cell response specific to B16 naturally expressed Trp2 TAA. CONCLUSION: The novel multi-drug daily metronomic chemotherapy evaluated in the present study was very effective in counterbalancing the immunosuppressive tumor microenvironment. Consequently, the intrinsic anti-tumor T cell immunity could exert its function, targeting specific TAA and significantly containing tumor growth. Overall, the results show that this represents a promising adjuvant approach to significantly enhance efficacy of intrinsic or vaccine-elicited tumor-specific cellular immunity.

Calreticulin acts as an adjuvant to promote dendritic cell maturation and enhances antigen-specific cytotoxic T lymphocyte responses against non-small cell lung cancer cells.

Dendritic cell (DC)-based immunotherapy has promising for treatment of non-small cell lung cancer (NSCLC). Melanoma-associated antigen 3 (MAGE-A3) is a tumor-specific antigen and expressed in approximately 35-40% of NSCLC tissues. Calreticulin (CALR) is a protein chaperone and can enhance DC maturation and antigen presentation. In this study, we evaluated the adjuvant activity of CALR in human DC maturation and their capacity to induce MAGE-A3-specific CD8+ cytotoxic T lymphocyte (CTL) responses to NSCLC in vitro. Infection with recombinant Ad-CALR and/or Ad-MAGE-A3, but not with control Ads, induced CALR and/or MAGE-A3 expression in DCs. Infection with Ad-CALR significantly increased the percentages of CD80+, CD83+, CD86+ and HLA-DR+ DCs and IL-12 secretion, but reduced IL-10 production in DCs. Co-culture of autologous lymphocytes with DC-Ad-CALR or DC-Ad-CM significantly increased the numbers of induced CD8+ CTLs. The percentages of IFNγ-secreting CTLs responding to SK-LU-1 and NCI-H522 NSCLC, but not to non-tumor NL-20 cells in Ad-C-CTL, Ad-M-CTL and Ad-CM-CTL were significantly higher than that of DC-CTL and Ad-null-CTL. Ad-C-CTL, Ad-M-CTL and Ad-CM-CTL, but not control DC-CTL and Ad-null-CTL, induced higher frequency of MAGE-A3+HLA-A2+ NCI-H-522 cell apoptosis, but did not affect the survival of MAGE-A3+HLA-A2- SK-LU-1 and non-tumor NL20 cells in vitro. Treatment with anti-HLA-I antibody, but not with anti-HLA-II, dramatically diminished the cytotoxicity of Ad-CM-CTLs against NCI-H522 cells. Our data indicated that CALR acted as an adjuvant to promote DC maturation, which induced CTL development and enhanced MAGE-A3-specific CTL cytotoxicity against NSCLC.

Antioxidant and immunomodulatory properties of polysaccharides from Allanblackia floribunda Oliv stem bark and Chromolaena odorata (L.) King and H.E. Robins leaves.

BACKGROUND: Many plant polysaccharides have shown high antioxidant and immunostimulating properties and can be explored as novel molecules with biological properties that can potentially improve immune function. The objective of this work was to characterize soluble and cell wall polysaccharides isolated from the stem bark of Allanblackia floribunda and Chromolaena odorata leaves and to evaluate their antioxidant and immunomodulatory properties. METHODS: Three polysaccharide fractions: soluble polysaccharides (PoS), pectins (Pec) and hemicelluloses (Hem) were extracted from A. floribunda stem bark and C. odorata leaves. These samples were analysed for their proteins, phenolic compounds and total sugar contents. The monosaccharide composition was determined by gas chromatography and arabinogalactan proteins content in PoS was evaluated by rocket electrophoresis. The in vitro antioxidant activities were evaluated by 1, 1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azino-bis-3-éthylbenzylthiazoline-6-sulphonic acid (ABTS) radical scavenging assays and ferrous ions chelating activity. Immunomodulatory activities were performed on the peripheral blood mononuclear cells (PBMCs) using proliferation and enzyme linked immunospot (ELISPOT) method to determine the production of an interferon-gamma. RESULTS: The characterization of the various fractions showed varied metabolites in each plant. In PoS fractions, Ara and Gal were the major monosaccharides found, indicating that arabinogalactans are the primary macromolecules. Hem fractions contained predominantly Xyl and GalA for A. floribunda and Xyl (upto 80 %) for and C. odorata. A. floribunda Hem fraction and C. odorata PoS fraction showed significant DPPH and ABTS radical scavenging activities and immunostimulatory activity via stimulation of PBMC and production of IFN-γ in a dose-dependent manner. CONCLUSION: The results obtained from this study support the ethnomedicinal use of the stem bark of A. floribunda and leaves of C. odorata. Further research is necessary to have supporting evidence that the antioxidative and immunomodulative activities of these fractions are really connected to the polysaccharides and not polyphenols.

Immunisation of Sheep with Bovine Viral Diarrhoea Virus, E2 Protein Using a Freeze-Dried Hollow Silica Mesoporous Nanoparticle Formulation.

Bovine viral diarrhoea virus 1 (BVDV-1) is arguably the most important viral disease of cattle. It is associated with reproductive, respiratory and chronic diseases in cattle across the world. In this study we have investigated the capacity of the major immunological determinant of BVDV-1, the E2 protein combined with hollow type mesoporous silica nanoparticles with surface amino functionalisation (HMSA), to stimulate immune responses in sheep. The current work also investigated the immunogenicity of the E2 nanoformulation before and after freeze-drying processes. The optimal excipient formulation for freeze-drying of the E2 nanoformulation was determined to be 5% trehalose and 1% glycine. This excipient formulation preserved both the E2 protein integrity and HMSA particle structure. Sheep were immunised three times at three week intervals by subcutaneous injection with 500 µg E2 adsorbed to 6.2 mg HMSA as either a non-freeze-dried or freeze-dried nanoformulation. The capacity of both nanovaccine formulations to generate humoral (antibody) and cell-mediated responses in sheep were compared to the responses in sheep immunisation with Opti-E2 (500 µg) together with the conventional adjuvant Quil-A (1 mg), a saponin from the Molina tree (Quillaja saponira). The level of the antibody responses detected to both the non-freeze-dried and freeze-dried Opti-E2/HMSA nanoformulations were similar to those obtained for Opti-E2 plus Quil-A, demonstrating the E2 nanoformulations were immunogenic in a large animal, and freeze-drying did not affect the immunogenicity of the E2 antigen. Importantly, it was demonstrated that the long term cell-mediated immune responses were detectable up to four months after immunisation. The cell-mediated immune responses were consistently high in all sheep immunised with the freeze-dried Opti-E2/HMSA nanovaccine formulation (>2,290 SFU/million cells) compared to the non-freeze-dried nanovaccine formulation (213-500 SFU/million cells). This study is the first to demonstrate that a freeze-dried silica mesoporous nanovaccine formulation gives balanced immune responses in a production animal.

Immune adjuvant effect of Juzentaihoto, a Japanese traditional herbal medicine, on tumor vaccine therapy in a mouse model.

Japanese traditional herbal medicine (Kampo) have been used to improve the general physical condition after surgery and to mitigate the side effects of radiation and chemotherapy in tumor patients. Juzentaihoto (JTT) consists of ten medical herbs, and is also called Shi-Quan-Da-Bu-Tang in Chinese herbal medicine. Among Kampo medicines, JTT has especially gained attention as a biological response modifier. Currently, clinical trials of various tumor vaccine therapies are being performed world-wide. However, tumor antigens that are inoculated as vaccines do not have high immunogenicity; thus, it is difficult to obtain an effective therapeutic effect. Thus, it is necessary to develop a tumor vaccine adjuvant that is more potent and very safe. In the present study, we examined the efficacy of JTT as an oral adjuvant when given together with tumor vaccines. As a result, JTT enhanced the phagocytic ability of OVA antigen and the presentation ability of OVA antigen in dendritic cells in vitro. Furthermore, tumor growth was markedly decreased, and the survival period was significantly prolonged in mice inoculated with mouse lymphoma, which is expressed with tumor model antigen. In conclusion, these findings suggest that JTT can be used with tumor vaccines as an immune adjuvant.

Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges.

Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.

Diagnostic value of enzyme-linked immunospot assay using CFP10/ESAT6 fusion protein as antigen in spinal tuberculosis.

OBJECTIVE: To establish a method of detecting spinal tuberculosis (TB) infection by enzyme-linked immunospot (ELlSPOT) assay and evaluate the value of CFP10/ESAT6 fusion protein for diagnosis of spinal TB. METHODS: Suspected spinal TB patients were prospectively recruited in two hospitals (First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine; Nanfang Hospital, Southern Medical University) from May 2012 to December 2013. Data on clinical characteristics of the patients and conventional laboratory results were collected. Compare and analyze the positive detection rate in spinal TB diagnosis by different methods including ELISPOT detection and conventional detection methods. RESULTS: 47 patients with spinal TB had available biopsy or surgical specimens for histopathological examination and 41 specimens had pathological features consistent with a diagnosis of TB infection. Among the spinal TB patients and non-TB disease patients,the overall sensitivity, specificity, positive predictive value, and negative predictive value of the ELISPOT assay in spinal TB diagnosis were 82.7%,87.2%,89.6%, and 79.1%,respectively; the 4 indexes of the PPD skin test were 61.5%, 46.2%, 60.4%, and 47.4%, respectively;those of the antibody detection were 55.8%, 61.5%, 65.9%, and 51.1%. The positive rate of ELISPOT was significantly higher than those of PPD skin test and antibody detection test (82.7% vs. 61.5%, Χ² =5.786, P=0.016; 82.7% vs. 55.8%, Χ² =8.847, P=0.003), but not significantly different from the positive rate of pathological examination (82.7% vs. 87.2%, Χ² =0.396, P=0.529). Moderate agreement was found between pathological examination and the ELISPOT assay (87.2%, Κ=0.498, P=0.001). CONCLUSION: With high sensitivity and specificity, the ELISPOT assay using CFP10/ESAT6 fusion protein as antigen is an effective technique for auxiliary diagnosis of spinal TB.

Diagnostic value of enzyme-linked immunospot assay using CFP10/ESAT6 fusion protein as antigen in spinal tuberculosis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a method of detecting spinal tuberculosis (TB) infection by enzyme-linked immunospot (ELlSPOT) assay and evaluate the value of CFP10/ESAT6 fusion protein for diagnosis of spinal TB.</p><p><b>METHODS</b>Suspected spinal TB patients were prospectively recruited in two hospitals (First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine; Nanfang Hospital, Southern Medical University) from May 2012 to December 2013. Data on clinical characteristics of the patients and conventional laboratory results were collected. Compare and analyze the positive detection rate in spinal TB diagnosis by different methods including ELISPOT detection and conventional detection methods.</p><p><b>RESULTS</b>47 patients with spinal TB had available biopsy or surgical specimens for histopathological examination and 41 specimens had pathological features consistent with a diagnosis of TB infection. Among the spinal TB patients and non-TB disease patients,the overall sensitivity, specificity, positive predictive value, and negative predictive value of the ELISPOT assay in spinal TB diagnosis were 82.7%,87.2%,89.6%, and 79.1%,respectively; the 4 indexes of the PPD skin test were 61.5%, 46.2%, 60.4%, and 47.4%, respectively;those of the antibody detection were 55.8%, 61.5%, 65.9%, and 51.1%. The positive rate of ELISPOT was significantly higher than those of PPD skin test and antibody detection test (82.7% vs. 61.5%, Χ² =5.786, P=0.016; 82.7% vs. 55.8%, Χ² =8.847, P=0.003), but not significantly different from the positive rate of pathological examination (82.7% vs. 87.2%, Χ² =0.396, P=0.529). Moderate agreement was found between pathological examination and the ELISPOT assay (87.2%, Κ=0.498, P=0.001).</p><p><b>CONCLUSION</b>With high sensitivity and specificity, the ELISPOT assay using CFP10/ESAT6 fusion protein as antigen is an effective technique for auxiliary diagnosis of spinal TB.</p>

Measuring T-cell responses against LCV and CMV in cynomolgus macaques using ELISPOT: potential application to non-clinical testing of immunomodulatory therapeutics.

A number of immunomodulatory therapeutics increase the risk of disease associated with latent herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), a member of the lymphocryptovirus (LCV) family that infects humans. The diseases associated with loss of immunity to these viruses can have major impacts on patients as well as on the commercial viability of the immunomodulatory therapeutics. In an effort to develop non-clinical methods for measuring effects on anti-viral immunity, we have developed an interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) assay to quantify the number of CMV or LCV-reactive T-cells in peripheral blood of cynomolgus macaques. After optimization of various parameters, the IFN-γ ELISPOT assay was characterized for specificity, intra-assay, monkey-to-monkey, and longitudinal variability and sensitivity to immunosuppression. The results show that nearly all animals have detectable responses against both CMV and LCV and responses were derived from T-cells specific to the virus of interest. Analyses of variability show assay reproducibility (≤23% CV), and that variability over time in anti-viral responses in individual animals (larger for LCV than for CMV) was ∼2-fold in most animals over a 3-month time period, which is predicted to allow for detection of drug-induced changes when using group sizes typical of non-clinical studies. In addition, the IFN-γ ELISPOT assay was capable of detecting decreases in the numbers of CMV and LCV reactive T-cells induced by immunosuppressive drugs in vitro. This assay may allow for non-clinical assessment of the effects of immunomodulatory therapeutics on anti-viral T-cell immunity in monkeys, and may help determine if therapeutics increase the risk of reactivating latent viral infections.

[Detection and molecular characterization of Potato virus S (PVS, Carlavirus) from Colombia]. / Detección serológica y caracterización molecular de Potato virus S (PVS, Carlavirus) en cultivos de papa de Colombia.

In Colombia, potato crops are affected by a wide variety of viruses such as PVY, PLRV, PVX, PMTV and PVS. Unfortunately, there are very few studies on the biology, distribution and pathogenicity of these viruses; this situation is even worse for the latent virus PVS. In this work, we evaluated the presence ofPVS in four Colombian provinces (Antioquia, Boyacá, Cundinamarca, Nariño) by the use of ELISA. We also studied the degree of molecular variation by sequence comparison of a segment of the gene encoding for the viral coat protein. In average, PVS was detected in 40% of 320 analyzed samples of potato leaves; the highest levels were observed in the East ofAntioquia (49%) and Pasto (Nariño) (47%), while in the other regions ranged between 35% and 42%. Analysis of sequence revealed the presence of two PVS strains in Colombia: three isolates were associated to PVSo (Ordinary) and twelve belonged to PVSA (Andean). A high diversity was observed among PVSA strains with percent identities in the range of 88-99%. These findings highlight the importance of strengthening seed certification programs and quarantine measures in Colombia for viruses like PVS, which can cause losses of up to 20% in potato crops and even higher in mixed virus infection.

Detección serológica y caracterización molecular de Potato virus S (PVS, Carlavirus) en cultivos de papa de Colombia / Detection and molecular characterization of Potato virus S (PVS, Carlavirus) from Colombia

In Colombia, potato crops are affected by a wide variety of viruses such as PVY, PLRV, PVX, PMTV and PVS. Unfortunately, there are very few studies on the biology, distribution and pathogenicity of these viruses; this situation is even worse for the latent virus PVS. In this work, we evaluated the presence of PVS in four Colombian provinces (Antioquia, Boyacá, Cundinamarca, Nariño) by the use of ELISA. We also studied the degree of molecular variation by sequence comparison of a segment of the gene encoding for the viral coat protein. In average, PVS was detected in 40% of 320 analyzed samples of potato leaves; the highest levels were observed in the East of Antioquia (49%) and Pasto (Nariño) (47%), while in the other regions ranged between 35% and 42%. Analysis of sequence revealed the presence of two PVS strains in Colombia: three isolates were associated to PVS O (Ordinary) and twelve belonged to PVS A (Andean). A high diversity was observed among PVS A strains with percent identities in the range of 88-99%. These findings highlight the importance of strengthening seed certification programs and quarantine measures in Colombia for viruses like PVS, which can cause losses of up to 20% in potato crops and even higher in mixed virus infection.

El cultivo de papa en Colombia es afectado por diversos virus, que incluyen PVY, PLRV, PVX, PMTV y PVS; aunque se han realizado pocos estudios sobre la biología, distribución y patogenicidad de dichos virus en Colombia, siendo especialmente escasa la información referente al PVS. En este trabajo se evaluó mediante pruebas de ELISA, la presencia del PVS en cuatro departamentos de Colombia, así como sus niveles de variación, a partir de la secuenciación de una porción del gen de la cápside viral. Los resultados indicaron una detección promedio del virus en el 40% de las 320 muestras analizadas, con zonas como el Oriente cercano de Antioquia (49%) y Pasto (Nariño) (47%), donde se detectó en mayor proporción el virus. Los análisis de variación molecular indicaron la presencia de las dos razas de PVS (Ordinaria y Andina) en Colombia, siendo los aislamientos de PVS A los más diversos, al pre- sentar un rango de identidad del 88 al 99%. Estos hallazgos indican que es imperativo el fortalecimiento de los programas de certificación de semilla y vigilancia cuarentenaria en el país, especialmente para virus como el PVS, que aunque puede ser asintomático, causa pérdidas hasta del 20% en cultivos de papa.

Development and preclinical safety evaluation of a new therapeutic HIV-1 vaccine based on 18 T-cell minimal epitope peptides applying a novel cationic adjuvant CAF01.

Therapeutic immunization of HIV-1-infected individuals with or without anti-retroviral therapy is a new promising disease prevention. To induce a new cytotoxic T(CD8) lymphocyte (CTL) immunity during chronic HIV-1 infection 15 infrequently targeted but conserved HLA-supertype binding CTL epitopes from Gag, Pol, Nef, Env, Vpu and Vif were identified. The 15 T(CD8) and three T(CD4) helper peptides were GMP synthesised and formulated with a new adjuvant CAF01 which is a synthetic two-component liposomic adjuvant comprising the quaternary ammonium dimethyl-dioctadecyl-ammonium (DDA) and the immune modulator trehalose 6,6'-dibehenate (TDB). Using IFN-γ ELISPOT assay, T-cell immune induction by the vaccine was found to both CD4 and CD8 T-cell restricted peptides in HLA-A2 transgenic mice. Comprehensive toxicity studies of the CAF01 adjuvant-alone and together with different vaccines showed that CAF01 when tested at human dose levels was safe and well tolerated with only local inflammation at the site of injection and no systemic reactions. No pharmacological safety issues were observed in Beagle dogs. The HIV-1 vaccine toxicity study in the Göttingen Minipig(®) showed no systemic toxicity from five repetitive i.m. injections, each with a 2-week interval, of either the 18 HIV-1 peptide antigen solution (AFO18) or the AFO18-CAF01, in which the 18 HIV-1 peptides were formulated with the CAF01 adjuvant. Distinct inflammatory responses were observed in the injected muscles of the AFO18-CAF01 vaccine treated animals as a result of the immune stimulating effect of the adjuvant on the vaccine. The results of the toxicity studies provide optimism for phase I clinical trials evaluating the therapeutic HIV-1 T-cell vaccination approach using multiple subdominant minimal epitope peptides applying the novel cationic adjuvant CAF01.

T cell monitoring of chemotherapy in experimental rat tuberculosis.

Mycobacterium tuberculosis is the causative agent of a pulmonary epidemic that is estimated to infect one-third of the world's population and that has an increased incidence of multidrug resistance. The evaluation of new chemical entities against M. tuberculosis is hampered by the lack of biological tools to help predict efficacy, from early drug development to clinical trials. As the rat is the animal species of choice in the pharmaceutical industry, we have developed a rat model of acute and chronic phases of M. tuberculosis infection for drug efficacy testing. In this model, we have evaluated the impact of tuberculosis drugs on T cell response using the enzyme-linked immunospot assay methodology. Infected rats treated with isoniazid (INH) or rifampin (RIF) responded to therapy, the potency of which was comparable to that seen in the mouse. Peripheral blood mononuclear cells from infected rats produced gamma interferon (IFN-γ) in response to RD-1 antigens, such as the 6-kDa early secretory antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). A decrease in IFN-γ spot-forming cells (SFCs) was consistently observed in response to drug treatment. In both the acute- and chronic-phase models, the T cell response was more sensitive to ESAT-6 than to CFP-10. The SFC count in response to ESAT-6 appears to be an indicator of bacterial killing in the rat. Collectively, our data suggest that the ESAT-6 response could be used as a potential surrogate of drug efficacy in the rat and that such a readout could help shorten drug testing during preclinical development.