Impact of Background Fruit Odors on Attraction of Drosophila suzukii (Diptera: Drosophilidae) to Its Symbiotic Yeast.

Background odors produced by plants in the environment can interfere with the response of insects to a point-releasing attractant, especially when their compositions overlap. In this study, a series of binary choice tests was conducted in a wind tunnel to investigate whether background odors emitted from cherry, blueberry, blackberry, or raspberry fruits would affect the level of Drosophila suzukii (Matsumura) attraction to its symbiotic yeast, Hanseniaspora uvarum (Niehaus) (Saccharomycetales: Saccharomycetaceae). Whether an increase in the intensity of background odors would affect the attractiveness of H. uvarum to D. suzukii was also investigated, either by increasing the number of cherry or raspberry fruit per cup or by increasing the number of fruit cups surrounding the cup baited with the yeast. In wind tunnel assays, background fruit odors interfering with D. suzukii attraction to the yeast varied among fruit types. Raspberry odor inhibited the attractiveness of H. uvarum to the fly the most, followed by blackberry odor, whereas cherry and blueberry odors had no significant impact on the attraction. An increase in the intensity of odors by adding more cherry or raspberry fruit per cup did not increase the impact of fruit odor on the attraction; however, adding more raspberry cups around H. uvarum linearly decreased its attractiveness, suggesting that background host fruit abundance and likely increase in host odor may influence D. suzukii attraction to yeast odor depending on host species.

Antigen-specific secretion of IFNγ and CXCL10 in whole blood assay detects Mycobacterium leprae infection but does not discriminate asymptomatic infection from symptomatic leprosy.

To advance toward a whole blood assay (WBA)-based test capable of facilitating the diagnosis of paucibacillary (PB) leprosy, we evaluated a prototype in-tube WBA using combinations of Mycobacterium leprae antigens. Blood was collected from newly diagnosed untreated PB (n=38), multibacillary (MB) (n=30), healthy household contacts (HHC) of MB (n=27), and endemic controls (n=61) residing in Goiânia and Fortaleza, Brazil. Blood was incubated with M. leprae cell sonicate, recombinant proteins (46f+LID-1; ML0276+LID-1), or controls (phosphate-buffered saline, phytohemagglutinin, M. tuberculosis purified protein derivative). Antigen-specific IFNγ production was observed in 71-84% and 55% of PB and HHC, respectively. Antigen-specific CXCL10 levels were similarly assessed to determine if, unlike IFNγ, CXCL10 could differentiate PB from HHC with repeated exposure/asymptomatic M. leprae infection. The CXCL10 levels induced in response to M. leprae antigens could not, however, differentiate PB from HHC. Despite these limitations, the WBAs reported here still represent important tools for assessing M. leprae infection rates and evaluating the impact of control measures.

Association of viable Mycobacterium leprae with Type 1 reaction in leprosy.

UNLABELLED: The working hypothesis is that, viable Mycobacterium leprae (M. leprae) play a crucial role in the precipitation of Type 1 reaction (T1R) in leprosy. MATERIAL AND METHODS: A total of 165 new multibacillary patients were studied. To demonstrate presence of viable M. leprae in reactional lesion (T1R+), three tests were used concurrently viz. growth in the mouse foot pad (MFP), immunohistochemical detection of M. leprae secretory protein Ag85, and 16s rRNA--using in situ RT-PCR. Mirror biopsies and non reactional lesions served as controls (T1R-). FINDINGS: A significantly higher proportion of lesion biopsy homogenates obtained at onset, from T1R(+) cases have shown unequivocal growth in MFP, proving the presence of viable bacteria, as compared to T1R(-) (P < 0.005). In contrast, few Mirror biopsies were positive in both T1R(+) and T1R(-). With respect to Ag85, while the overall positivity was higher in T1R(+) (74%), however the intensity of staining (Grade 2+) was disproportionately higher in T1R(+) BT-BB lesions 11/20 (55%). In the rebiopsies obtained during a repeat episode of T1R, Ag 85 as well as 16s rRNA, positivity (62% & 100%) was higher in T1R(+). It is inferred therefore 'viable' bacteria are an essential component in T1R and difference in the quality of bacilli, not the quantity or the ratio of dead to viable play a role in the precipitation of T1R. In conclusion, the findings show that 'metabolically active' M. leprae is a component/prerequisite and the secretory protein Ag 85, might be the trigger for precipitation of T1R.

Mutation at codon 442 in the rpoB gene of Mycobacterium leprae does not confer resistance to rifampicin.

BACKGROUND: Rifampicin is the major drug in the treatment of leprosy. The rifampicin resistance of Mycobacterium leprae results from a mutation in the rpoB gene, encoding the ß subunit of RNA polymerase. As M. leprae is a non-cultivable organism observation of its growth using mouse food-pad (MFP) is the only Gold Standard assay used for confirmation of "in-vivo" drug resistance. OBJECTIVE: Any mutation at molecular level has to be verified by MFP assay for final confirmation of drug resistance in leprosy. MATERIAL AND METHODS: In the present study, M. leprae strains showing a mutation only at codon 442 Gln-His and along with mutation either at codon 424 Val-Gly or at 438 Gln-Val within the Rifampicin Resistance Determining Region (RRDR) confirmed by DNA sequencing and by high resolution melting (HRM) analysis were subjected for its growth in MFP. RESULT AND CONCLUSION: The M. leprae strain having the new mutation at codon 442 Gln-His was found to be sensitive to all the three drugs and strains having additional mutations at 424 Val-Gly and 438 Gln-Val were conferring resistance with Multi drug therapy (MDT) in MFP. These results indicate that MFP is the gold standard method for confirming the mutations detected by molecular techniques.

Cytotoxicity potentials of eleven Bangladeshi medicinal plants.

Various forms of cancer are rising all over the world, requiring newer therapy. The quest of anticancer drugs both from natural and synthetic sources is the demand of time. In this study, fourteen extracts of different parts of eleven Bangladeshi medicinal plants which have been traditionally used for the treatment of different types of carcinoma, tumor, leprosy, and diseases associated with cancer were evaluated for their cytotoxicity for the first time. Extraction was conceded using methanol. Phytochemical groups like reducing sugars, tannins, saponins, steroids, gums, flavonoids, and alkaloids were tested using standard chromogenic reagents. Plants were evaluated for cytotoxicity by brine shrimp lethality bioassay using Artemia salina comparing with standard anticancer drug vincristine sulphate. All the extracts showed potent to moderate cytotoxicity ranging from LC50 2 to 115 µg/mL. The highest toxicity was shown by Hygrophila spinosa seeds (LC50 = 2.93 µg/mL) and the lowest by Litsea glutinosa leaves (LC50 = 114.71 µg/mL) in comparison with standard vincristine sulphate (LC50 = 2.04 µg/mL). Among the plants, the plants traditionally used in different cancer and microbial treatments showed highest cytotoxicity. The results support their ethnomedicinal uses and require advanced investigation to elucidate responsible compounds as well as their mode of action.

Lateral flow assay for simultaneous detection of cellular- and humoral immune responses.

OBJECTIVE: The development of a cytokine detection assay suitable for detection of multiple biomarkers for improved diagnosis of mycobacterial diseases. DESIGN AND METHODS: A lateral flow (LF) assay to detect IL-10 was developed utilizing the up-converting phosphor (UCP) reporter-technology. The assay was evaluated using blood samples of leprosy patients. Multiplex applications were explored targeting: 1) IL-10 and IFN-γ in assay buffer; 2) IL-10 and anti-phenolic glycolipid (PGL-I) antibodies in serum from leprosy patients. RESULTS: Detection of IL-10 below the targeted level of 100pg/mL in serum was shown. Comparison with ELISA showed a quantitative correlation with R(2) value of 0.92. Multiplexing of cytokines and simultaneous detection of cytokine and antibody was demonstrated. CONCLUSIONS: The UCP-LF IL-10 assay is a user-friendly, rapid alternative for IL-10 ELISAs, suitable for multiplex detection of different cytokines and can be merged with antibody-detection assays to simultaneously detect cellular- and humoral immunity.

A user-friendly, highly sensitive assay to detect the IFN-gamma secretion by T cells.

OBJECTIVES: Development of a user-friendly test alternative to ELISA-based assays to detect IFN-gamma by in vitro cultured peripheral blood mononuclear cells (PBMC) stimulated with pathogen-derived antigens. DESIGN AND METHODS: The molecular components of an operational IFN-gamma ELISA-based test were applied in a lateral flow (LF) immuno-sandwich assay using up-converting phosphor (UCP) reporter particles. The analytical sensitivity of the UCP-LF IFN-gamma assay (ULIGA) was determined and the assay was qualitatively validated with a selection of 60 supernatants derived from PBMC cultures stimulated with M. leprae derived antigens, mitogen or medium alone. RESULTS: ULIGA indicated an analytical sensitivity better than 2 pg/mL, and demonstrated four orders of magnitude dynamic range. The assay correlated well with the IFN-gamma ELISA. CONCLUSIONS: ULIGA allows detection well below the cutoff value (100 pg/mL) used to define positive responses in the IFN-gamma ELISA. The test procedure is less demanding in respect to equipment and labor, and is suited for testing single samples.

Reverse line probe assay for the rapid detection of rifampicin resistance in Mycobacterium leprae.

Mutations in the rpoB gene of 40 biopsy isolates of Mycobacterium leprae were analyzed by reverse hybridization-based line probe assay after PCR, and nine distinct single-nucleotide substitutions were found. Among them, a 3-nucleotide substitution was found in two, and 2-nucleotide substitutions were found in seven isolates. This is a new finding of multiple mutations in a single point of the rpoB gene for rifampicin resistance. This investigation demonstrates that the pattern of mutations in the rpoB gene for rifampicin resistance in Nepal involves more variety.

10-12 years follow-up of highly bacillated BL/LL leprosy patients on combined chemotherapy and immunotherapy.

This study reports the follow-up results of 36 highly bacillated untreated BL/LL cases who were serially allocated to three treatment groups. Group I patients received a modified WHO regimen (Rifampicin 600 mg once a month supervised, 50 mg of Clofazimine and 100 mg of Dapsone daily unsupervised) and BCG 0.1 mg per dose 6 monthly; group II patients received the same multi-drug treatment (MDT) and Mw (2 x 10(8) killed bacilli per dose) 6 monthly: group III patients received the same MDT with 0.1 ml of distilled water 6 monthly and acted as a control. Treatment was continued till smear negativity. All these three groups were comparable by their initial clinical score, bacteriological index (BI), viable bacilli as assessed by the mouse foot pad (MFP), bacillary adenosine triphosphate (ATP) content and also histologically at the time of starting treatment. All these parameters were evaluated every 6 months. The vaccines were well tolerated. All the patients in group I became smear negative by 3.5 years, in group II in 3 years whereas those in group III took 5 years. The incidence of reactions was the same in all the groups during the first 2 years, however, patients of group III (MDT + placebo) continued to have reactions up to 3 years. No viable bacilli could be detected in the local and distal sites as estimated by MFP and bacillary ATP after 12 months in both the immunotherapy groups. These could be detected in patients on MDT alone up to 24 months of therapy. Histologically patients in both the immunotherapy groups (groups I and II) showed accelerated granuloma clearance, histological upgrading and non-specific healing without granuloma formation both at the local and distal sites and this was achieved much earlier compared to the MDT + placebo group. Thus, by the addition of immunotherapy the effective treatment period of achieving bacteriological negativity could be reduced by about 40%, time period of reactions reduced by 33% and there were no reactions and/or relapses in the 10-12 years post-treatment follow-up.

Detection of viable organisms in leprosy patients treated with multidrug therapy.

Cutaneous biopsies were collected from multibacillary leprosy patients who attended the out-patient department of Jalma Institute for treatment at different time intervals, i.e. 6 months, 12 months, 18 months, 24 months, 30 months, 36 months and 42 months after starting multidrug therapy (MDT) when they were still skin smear positive. Biopsies were processed for inoculation into mouse foot pad (MFP) and estimation of bacillary ATP levels by bioluminescent assay (ATP assay) by earlier established procedures. Viable bacilli were detectable after 1 year (25% cases by MFP and 31% cases by ATP assay), 2 years (8% cases by MFP and 12% cases by ATP assay) and 3 years (4% cases by both MFP and ATP assays). Overall, the percentage of the persisters was 10% by MFP and 13% by ATP assay. It would be important to carry out surveillance studies in larger number of BL/LL cases to know the trends and also the resultant relapses.

Nasal mucosa and skin of smear-positive leprosy patients after 24 months of fixed duration MDT: histopathological and microbiological study.

The skin and nasal mucosa of 10 lepromatous leprosy patients who had completed 24 doses of fixed duration multidrug therapy (MDT) but who continued to be skin-smear positive for acid-fast bacilli (AFB) were examined histopathologically. The nasal mucosa showed granuloma fractions that exceeded those seen in the skin specimens, signifying that activity in this region subsides much more gradually than the activity in the skin. Mouse foot pad studies done using T900r mice with an inoculum from the nasal mucosa biopsy specimens of these patients did not demonstrate any growth of Mycobacterium leprae, indicating that these bacilli were not viable. A skin specimen from one patient grew significant amounts of bacteria in the T900r mouse foot pad. These results show that 2 years of treatment with MDT would prevent dissemination of M. leprae from the nasal mucosa and, therefore, should preclude further transmission of the disease. It also indicates that viable bacteria might persist in the skin of patients, especially those with an initial bacterial index of > or = 4+ who have completed 24 doses of regular MDT. Therefore, a more cautious approach to administering only 12 doses of MDT to highly positive multibacillary patients is suggested.

Differential development of CD4 and CD8 cytotoxic T cells (CTL) in PBMC across the leprosy spectrum; IL-6 with IFN-gamma or IL-2 generate CTL in multibacillary patients.

In the present study we evaluated the contribution of CD4 and CD8 T cells on the antigen-specific cytotoxic activity induced by whole Mycobacterium leprae in leprosy patients and normal controls (N) as well as the modulation of this activity by some cytokines. Peripheral blood mononuclear cells (PBMC) from N or from leprosy patients were stimulated with antigen in the presence or absence of cytokines for 7 days. M. leprae-stimulated PBMC were depleted of CD4 or CD8 antigen-bearing cells and employed as effector cells in a 4-hr [31Cr]-release assay against autologous M. leprae-pulsed macrophages. Our results demonstrate that both CD4 and CD8 T cells contribute to M. leprae-induced cytotoxic activity, with differences observed in paucibacillary (PB) and multibacillary (MB) patients. CD8-mediated cytotoxic activity is higher than that of CD4 cells in PB patients, while in MB patients CD4 cytotoxicity is predominant. Our data also demonstrate that the generation of CD4 and CD8 cytotoxic T lymphocytes (CTL) can be modulated differentially by interleukin-4 (IL-4), IL-6, gamma interferon (IFN-gamma), or IL-2. Although MB patients developed the lowest CTL response, cytokines such as IL-6 plus IL-2 or IFN-gamma were able to generate both CD4 and CD8 cytotoxic T cells from MB patients. In PB patients, IL-6 plus IFN-gamma displayed the highest stimulation on CD8 effector cells. Thus, an important role may be assigned to IL-6, together with IL-2 or IFN-gamma, in the differentiation of M. leprae-specific CTL effector cells.

Viability determination of M.leprae: comparison of normal mouse foot pad and bacillary ATP bioluminescence assay.

Correlation between viability assessment by mouse foot pad and ATP bioluminescence was studied in biopsy specimens from multibacillary leprosy cases. Biopsies were processed for inoculation into mouse foot pad and estimation of bacillary ATP levels by bioluminescent assay by earlier established procedures. ATP content as pg/million bacilli was estimated and correlation was assessed with growth in the mouse foot pad. It was observed that when the ATP content was > 36 pg/million bacterial cells, (> 1% probable viables) there was growth in the mouse foot pad from all the specimens. Similar results were observed when the ATP content was in the range of 3.6 to 35.99 pg/million cells (0.1 to 1% probable viables). The positivity rates in the mouse foot pad decreased when the ATP content decreased further. No positive growth in the specimens below 0.04 pg/million bacilli (< 0.001% viable organisms) was observed. These findings show an overall correlation between viability assessed by mouse foot pad and ATP bioluminescence. These observations validate the concept of ATP content of viable unit of M.leprae being in the order of 10(-15) g/live cell which is in the same order of magnitude as a colony forming unit of cultivable mycobacteria.

The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL.

Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.

Early detection of rifampin in human nerve tissue after an oral dose of 600 milligrams.

Rifampin in picogram quantities inhibited the ability of Mycobacterium bovis 44 BCG P3 to release 14CO2 from the oxidation of [14C]palmitic acid. By using these mycobacteria in a bioassay, samples of serum and posterior tibial nerve were assayed for inhibitory concentrations of rifampin. Within 8 to 12 h after ingestion of 600 mg of rifampin, the drug was detected in eight patients in concentrations ranging from 0.52 to 4.1 micrograms/ml in serum and in concentrations ranging from 0.6 to 6.3 ng/mg in posterior tibial nerve fiber tissue.

Methods for determining concentrations of antimicrobial agents in human monocytes.

Human monocytes can be derived from the leukocyte-rich by-product of donors' blood available after platelet separation. Large volumes of the monocyte samples obtained from this product provided an opportunity to conduct experiments with relatively high concentrations of the antimicrobial agents sufficient for their detection in bioassays, thus avoiding the necessity of working with the radiolabelled drugs. Washing of the cells after their exposure to the drug may lead to an extraction of the tested agent from the cell, especially if it is a substance of low molecular weight. In our experiments we excluded the washing step, and separated the monocytes from the extracellular medium by velocity gradient centrifugation. In experiments with two rifamycins, the cell pellet as well as the extracellular fluid were subjected to a bioassay using Micrococcus luteus as a target organism. The method showed good reproducibility and consistency in results obtained.

Linfocitos Th1, Th2 y enfermedad / Lymphocytes Th1, Th2 and disease

Al activarse, los linfocitos T CD4+ muestran una producción de citocinas que usualmente sigue tres patrones distintos y estereotipados; estos patrones son producidos por subpoblaciones discretas que se denominan Th1, Th2 y Th0. Las células Th1 secretan principalmente interferón- (IFN-), interleucina-2 (IL-2) y factor de necrosis tumoral (FNT), citocinas que tienen un papel clave en la respuesta inmune mediada por células y el fenómeno de hipersensibilidad de tipo tardío. Las células Th2 producen IL-4, IL-5, IL-6, IL-9, IL-10 e IL-13, las cuales juegan un papel clave en la generación de la respuesta inmune humoral. Existe una regulación negativa recíproca entre las células Th1 y Th2 ya que ciertas citocinas de las primeras (principalmente IFN-) y de las segundas (IL-4, IL-10) tienen un efecto inhibitorio sobre células Th2 y Th1 respectivamente. Las células Th0 producen citocinas tipo Th1 y Th2. Es posible determinar el patrón de citocinas que están siendo producidas en un tejido, lo que permite inferir si ocurre una activación preferencial de células Th1, Th2 o Th0 en ese sitio. La activación preferencial de subpoblaciones de linfocitos CD4+ tiene un papel importante en la patogenia de enfermedades infecciosas y autoinmunes. Los pacientes con lepra lepromatosa tienen una activación preferencial de células Th2, lo cual conduce a una enfermedad diseminada con ausencia de respuesta inmune celular. La activación preferencial de células Th2 puede ser de importancia en la patogenia de enfermedades autoinmunes generalizadas, en tanto que las células Th1 tienen importancia en padecimientos autoinmunes órgano-específicos. Resulta factible el manipular in vivo la activación preferencial de linfocitos Th1 y Th2, lo cual puede tener implicaciones terapéuticas importantes