B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Plasmodium products persist in the bone marrow and promote chronic bone loss.

Although malaria is a life-threatening disease with severe complications, most people develop partial immunity and suffer from mild symptoms. However, incomplete recovery from infection causes chronic illness, and little is known of the potential outcomes of this chronicity. We found that malaria causes bone loss and growth retardation as a result of chronic bone inflammation induced by Plasmodium products. Acute malaria infection severely suppresses bone homeostasis, but sustained accumulation of Plasmodium products in the bone marrow niche induces MyD88-dependent inflammatory responses in osteoclast and osteoblast precursors, leading to increased RANKL expression and overstimulation of osteoclastogenesis, favoring bone resorption. Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause bone loss. Supplementation of alfacalcidol, a vitamin D3 analog, could prevent the bone loss. These results highlight the risk of bone loss in malaria-infected patients and the potential benefits of coupling bone therapy with antimalarial treatment.

Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria.

Cerebral malaria is a complication of Plasmodium falciparum infection characterized by sudden coma, death, or neurodisability. Studies using a mouse model of experimental cerebral malaria (ECM) have indicated that blood-brain barrier disruption and CD8 T cell recruitment contribute to disease, but the spatiotemporal mechanisms are poorly understood. We show by ultra-high-field MRI and multiphoton microscopy that the olfactory bulb is physically and functionally damaged (loss of smell) by Plasmodium parasites during ECM. The trabecular small capillaries comprising the olfactory bulb show parasite accumulation and cell occlusion followed by microbleeding, events associated with high fever and cytokine storm. Specifically, the olfactory upregulates chemokine CCL21, and loss or functional blockade of its receptors CCR7 and CXCR3 results in decreased CD8 T cell activation and recruitment, respectively, as well as prolonged survival. Thus, early detection of olfaction loss and blockade of pathological cell recruitment may offer potential therapeutic strategies for ECM.

Lipocalin 2 bolsters innate and adaptive immune responses to blood-stage malaria infection by reinforcing host iron metabolism.

Plasmodium parasites multiply within host erythrocytes, which contain high levels of iron, and parasite egress from these cells results in iron release and host anemia. Although Plasmodium requires host iron for replication, how host iron homeostasis and responses to these fluxes affect Plasmodium infection are incompletely understood. We determined that Lipocalin 2 (Lcn2), a host protein that sequesters iron, is abundantly secreted during human (P. vivax) and mouse (P. yoeliiNL) blood-stage malaria infections and is essential to control P. yoeliiNL parasitemia, anemia, and host survival. During infection, Lcn2 bolsters both host macrophage function and granulocyte recruitment and limits reticulocytosis, or the expansion of immature erythrocytes, which are the preferred target cell of P. yoeliiNL. Additionally, a chronic iron imbalance due to Lcn2 deficiency results in impaired adaptive immune responses against Plasmodium parasites. Thus, Lcn2 exerts antiparasitic effects by maintaining iron homeostasis and promoting innate and adaptive immune responses.

Serologic markers in relation to parasite exposure history help to estimate transmission dynamics of Plasmodium vivax.

Plasmodium vivax infection has been gaining attention because of its re-emergence in several parts of the world. Southeastern Turkey is one of the places in which persistent focal malaria caused exclusively by P. vivax parasites occurs. Although control and elimination studies have been underway for many years, no detailed study has been conducted to understand the mechanisms underlying the ineffective control of malaria in this region. Here, for the first time, using serologic markers we try to extract as much information as possible in this region to get a glimpse of P. vivax transmission. We conducted a sero-immunological study, evaluating antibody responses of individuals living in Sanliurfa to four different P. vivax antigens; three blood-stage antigens (PvMSP119, PvAMA1-ecto, and PvSERA4) and one pre-erythrocytic stage antigen (PvCSP). The results suggest that a prior history of malaria infection and age can be determining factors for the levels and sustainability of naturally acquired antibodies. Significantly higher antibody responses to all the studied antigens were observed in blood smear-negative individuals with a prior history of malaria infection. Moreover, these individuals were significantly older than blood smear-negative individuals with no prior history of infection. These data from an area of sole P. vivax-endemic region may have important implications for the global malaria control/elimination programs and vaccine design.

The melanogenesis-inhibitory, anti-inflammatory, and chemopreventive effects of limonoids in n-hexane extract of Azadirachta indica A. Juss. (neem) seeds.

Seventeen limonoids (tetranortriterpenoids 1-17) were isolated from the n-hexane extract of Azadirachta indica (neem) seeds. The previously unidentified compound 16 was established by spectroscopy to be 17-defurano-17-oxosalannin. The effects of six compounds, 6 and 11-15, on melanogenesis in B16 melanoma cells was evaluated; 2 compounds, salannin (13) and 3-deacetylsalannin (15), exhibited marked inhibitory effects (70-74% reduction of melanin content at 25 µg/mL) with only minor cytotoxicity (79-85% of cell viability). Eleven compounds, 2, 3, 5, 6, and 9-15, were evaluated for inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1.7 nmol/ear) in mice; all exhibited marked anti-inflammatory activity (ID(50) values 0.22-0.57 µmol/ear). In addition, compounds 6 and 11-16 exerted moderate inhibition (IC(50) values of 410-471 mol ratio/32 pmol TPA) of TPA-induced Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells. The triacylglycerol fraction of the n-hexane extract contained oleic acid (50.2%) as the most predominant fatty acid constituent.

Three new cyclic diarylheptanoids and other phenolic compounds from the bark of Myrica rubra and their melanogenesis inhibitory and radical scavenging activities.

Ten cyclic diarylheptanoids (1-10), including three new compounds: myricanone 5-O-alpha-L-arabinofuranosyl-(1-->6)-beta-D-glucopyranoside (7), myricanone 17-O-beta-D-(6'-O-galloyl)-glucopyranoside (8), and 16-methoxy acerogenin B 9-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (10), along with two flavonoids (11, 12), were isolated from the extracts of Myrica rubra (Myricaceae) bark. The structures of new compounds were determined on the basis of spectroscopic methods. On evaluation of compounds 1-12 against the melanogenesis in the B16 melanoma cells, six compounds, 3, 5, 7, 8, 10, and 12, exhibited inhibitory effects with 30-56% reduction of melanin content at 25 microg/mL with no or very weak toxicity to the cells (82-103% of cell viability at 25 microg/mL). In addition, upon evaluation of compounds 1-12 against the scavenging activities of free radicals [against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical], seven compounds, 1, 3, 5, 6, 8, 11, and 12, showed potent scavenging activity [IC(50) 2-21 microM (0.6-7.3 microg/mL)].

Melanogenesis inhibitory, anti-inflammatory, and chemopreventive effects of limonoids from the seeds of Azadirachta indicia A. Juss. (neem).

Thirty-one nortriterpenoids, including 28 limonoids (1-28) and 3 degraded limonoids (29-31), and one diterpenoid (32), were isolated from the seed extract of Azadirachta indica (neem). Among these, six were new compounds and their structures were established to be 15-hydroxyazadiradione (3), 7-benzoyl-17-hydroxynimbocinol (5), 23-deoxyazadironolide (12), limocin E (13), 23-epilimocin E (14), and 7alpha-acetoxy-3-oxoisocopala-1,13-dien-15-oic acid (32). Upon evaluation of compounds 1-32 on the melanogenesis in the B16 melanoma cells, five compounds, 20, 26, 27, 29, and 31, exhibited marked inhibitory effect (74-91% reduction of melanin content at 25 microg/mL) with no or almost no toxicity to the cells. Seven compounds, 1, 6, 9, 10, 18, 20, and 26, on evaluation for their inhibitory effect against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 microg/ear) in mice, exhibited, except for compound 26, marked anti-inflammatory activity (ID(50) values 0.09-0.26 mg/ear). In addition, all of the 32 compounds exhibited moderate or potent inhibitory effects (IC(50) values of 230-501 mol ratio/32 pmol TPA) against the Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA. Furthermore, on evaluation of azadirachtin B (21) for its anti-tumor-initiating activity on the two-stage carcinogenesis of mouse skin tumor induced by peroxynitrite (ONOO-; PN) as an initiator and TPA as a promoter, this exhibited marked inhibitory activity.

Serodiagnostic contributions of antibody titers against mycobacterial lipid antigens in Mycobacterium avium complex pulmonary disease.

BACKGROUND: Although the incidence of pulmonary tuberculosis is decreasing, the number of immunocompetent patients with Mycobacterium avium complex (MAC) pulmonary disease is steadily increasing. Therefore, albeit not contagious, MAC pulmonary disease needs to be diagnosed rapidly and accurately. We examined the serodiagnostic contributions of serum immunoglobulin G antibody titers against the species-specific and -common mycobacterial lipid antigens in the diagnosis of MAC pulmonary disease. METHODS: Serum samples were obtained from 65 patients with MAC pulmonary disease, 15 patients with suspected MAC disease, 25 patients with pulmonary tuberculosis, 10 patients with Mycobacterium kansasii disease, and 100 healthy volunteers (control subjects). We measured the serum immunoglobulin G antibody titers against trehalose monomycolate (TMM-M) and apolar-glycopeptidolipid (GPL), lipid antigens extracted from MAC. RESULTS: In patients with MAC pulmonary disease, the antibody titers against TMM-M and apolar-GPL were significantly higher than those in the other patient groups or in the control subjects. By receiver operator characteristic curve analysis, an optical density of 0.27, corresponding to the optimal cutoff antibody titer against TMM-M, was associated with a sensitivity of 89.2% and a specificity of 97.0%, and an optical density of 0.33, corresponding to the optimal cutoff antibody titer against apolar-GPL, was associated with a sensitivity of 89.2% and a specificity of 94.0%. CONCLUSIONS: Measurements of antibody titers against TMM-M and apolar-GPL would be useful in the diagnosis of MAC pulmonary disease and in the differential diagnosis of mycobacterial pulmonary disease.

Comparable studies of immunostimulating activities in vitro among Mycobacterium bovis bacillus Calmette-Guérin (BCG) substrains.

During the serial passage of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in different countries after initial seed distribution from the Pasteur Institute, specific insertions and deletions in the genome among BCG substrains were observed and speculated to result in differences in immunological activities. 'Early-shared strains' of BCG (Russia, Moreau, Japan, Sweden, Birkhaug), distributed by the Pasteur Institute, which conserve three types of mycolate (alpha, methoxy, keto) in cell wall, exhibited stronger activities of induction of nitric oxide, interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-alpha, from human epithelial cell line A549, human myelomonocytic cell line THP-1 and mouse bone marrow cells in the presence of interferon-gamma (IFN-gamma) than did 'late-shared strains' of BCG (Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia, Pasteur). The stronger induction of IL-12 and TNF-alpha in the presence of IFN-gamma was also observed by trehalose 6,6'-dimycolate (TDM) extracted from BCG-Japan than by TDM from BCG-Connaught, which lacks the methoxymycolate residue. These results suggest that 'early-shared strains' are more potent immunostimulating agents than 'late-shared strains', which could be attributed partially to methoxymycolate. Our study provides the basic information for immunological characterization of various BCG strains and may contribute to a re-evaluation of them as a reference strain for vaccination against tuberculosis.

Human Th1 differentiation induced by lipoarabinomannan/lipomannan from Mycobacterium bovis BCG Tokyo-172.

Mycobacterium tuberculosis (tubercle bacilli) and the related acid-fast bacteria including Mycobacterium bovis Bacille Calmett-Guerin (BCG) have a characteristic cell wall (CW) containing various lipoglycans and glycolipids. Such lipoglycans have been reported to activate type-I inflammatory responses via dendritic cells (DCs) through Toll-like receptor 2. In this study, lipoglycans, lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinositol mannoside (PIM), were purified from the CW fractions of M. bovis BCG Tokyo-172, and the effect on the differentiation of human peripheral blood naive CD4 T cells into T(h)1 and T(h)2 was examined. LAM/LM molecules enhanced T(h)1 differentiation under both T(h)1 and T(h)2 conditions, whereas some other glycolipids and phospholipid enhanced T(h)2 differentiation under T(h)2 conditions. Other components had little effect under the given conditions. Even in highly purified CD4 T cell cultures, LAM/LM enhanced T(h)1 generation only under T(h)1 culture conditions. These results indicate that LAM/LM possesses a potent augmenting activity in T(h)1 differentiation in human CD4 T cells. LAM/LM appeared to act directly on naive CD4 T cells to enhance T(h)1 differentiation under T(h)1 culture conditions, while acting indirectly to up-regulate the generation of T(h)1 cells via IL-12/DCs under T(h)1 and T(h)2 conditions. Therefore, these results provide the first evidence indicating that LAM/LM from M. bovis BCG may possess a potent modulating activity in the human system, and thus supporting the strategy for the use of BCG components in the vaccine development for such T(h)2 diseases as allergic asthma and rhinitis.

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105).

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).

Identification of trehalose dimycolate (cord factor) in Mycobacterium leprae.

Glycolipids of Mycobacterium leprae obtained from armadillo tissue nodules infected with the bacteria were analyzed. Mass spectrometric analysis of the glycolipids indicated the presence of trehalose 6,6'-dimycolate (TDM) together with trehalose 6-monomycolate (TMM) and phenolic glycolipid-I (PGL-I). The analysis showed that M. leprae-derived TDM and TMM possessed both alpha- and keto-mycolates centering at C78 in the former and at C81 or 83 in the latter subclasses, respectively. For the first time, MALDI-TOF mass analyses showed the presence of TDM in M. leprae.

New packaging method of mycobacterial cell wall using octaarginine-modified liposomes: enhanced uptake by and immunostimulatory activity of dendritic cells.

Despite the potential of mycobacterial cell wall (CW) components to serve as immunotherapeutic agents, this application is hampered by the molecules' unfavorable physicochemical properties, such as its high molecular weight, poor solubility and negatively charged nature. Here we describe a new mycobacterial CW delivery system that uses an efficient and simple packaging method. This is achieved by incorporating mycobacterial CW into liposomes and attaching arginine octamers (R8) to the liposome surface. R8-modified liposomes improve the uptake of mycobacterial CW by dendritic cells (DC) and enhance its immunostimulatory activity. High R8 surface density promoted high levels of mycobacterial CW uptake by DC compared to low density R8-modified liposomes. Maturation markers (CD80, CD86, MHC Class II molecules) showed significantly enhanced expression on DC pulsed with high density R8-modified liposomes containing mycobacterial CW. Moreover, R8-modified liposomes with mycobacterial CW incorporated induced production of IL-12 p40 by DC, at levels similar to those produced by lipopolysaccharide-pulsed DC. We assert that R8-modified liposomes with mycobacterial CW incorporated should have tremendous potential as immune-potentiating agents.

Molecular and supra-molecular structure related differences in toxicity and granulomatogenic activity of mycobacterial cord factor in mice.

To establish the structure biological activity relationship of cord factor (trehalose 6,6'-dimycolate, TDM), we compared the molecular or supra-molecular structure of TDM micelles with toxicity, thymic atrophy and granulomatogenicity in lungs and spleen of BALB/c mice. According to the difference in the mycolyl subclass composition, TDM was divided into two groups, one possessing alpha-, methoxy- and keto-mycolates in M. tuberculosis H37Rv, M. bovis BCG and M. kansasii (group A) and the other having alpha-, keto- and wax ester-mycolates in M. avium serotype 4, M. phlei and M. flavescens (group B), although mycolic acid molecular species composition differed in each group considerably. Supra-molecular structure of TDM micelle differed species to species substantially and the micelle size of TDM from M. bovis BCG Connaught was the largest. The highest toxicity was shown with TDM from M. tuberculosis H37Rv which possessed the highest amount of alpha- (47.3%) and methoxy-mycolates (40.8%), while TDM from M. phlei having the low amount of alpha-mycolate (11.6%) showed almost no toxicity with the given doses. The thymic atrophy was observed with TDM from group A, but not with TDM from group B. On the other hand, TDM from group B showed massive lung granulomatogenic activity based on the histological observations and organ indices. Taken together, group A TDM showed a wide variety of micelle sizes and specific surface areas, high to low toxicity and marked to moderate granulomatogenicity, while group B TDM showed smaller sizes of micelles and larger specific surface areas, lower toxicity but higher granulomatogenicity in lungs. Existence of higher amount of longer chain alpha-mycolates in TDM appeared to be essential for high toxicity and thymic apoptotic activity, whereas TDM possessing wax ester-mycolate with smaller sized micelles seemed to be less toxic, but more granulomatogenic in lungs in mice. Thus, the mycolic acid subclass and molecular species composition of TDM affect critically the micelle forms, toxicity and granulomatogenicity in mice, while the relative abundances and carbon chain length of alpha-mycolate affected the toxicity in mice.

Mycobacterial sulfolipid shows a virulence by inhibiting cord factor induced granuloma formation and TNF-alpha release.

Virulence mechanism of infection with Mycobacterium tuberculosis is currently focused to be clarified in the context of cell surface lipid molecule. Comparing two mycobacterial glycolipids, we observed toxicity and prominent granulomatogenic activity of trehalose 6,6'-dimycolate (TDM) injection in mice, evident by delayed body weight gain and histological observations, whereas 2,3,6,6'-tetraacyl trehalose 2'-sulfate (SL) was non-toxic and non-granulomatogenic. Likewise, TDM but not SL caused temporarily, but marked increase of lung indices, indicative of massive granuloma formation. Interestingly, co-administration of TDM and SL prevented these symptoms distinctively and SL inhibited TDM-induced release of tumor necrosis factor alpha (TNF-alpha) in a dose-dependent manner. Histological findings and organ index changes also showed marked inhibition of TDM induced granuloma formation by co-administration of SL. Simultaneous injection of SL together with TDM was highly effective for this protection, as neither injection 1h before nor after TDM injection showed highly inhibitory. In parallel studies on a cellular level, TDM elicited strong TNF-alpha release from alveolar but not from peritoneal macrophages in vitro. This effect was blocked when alveolar macrophages were incubated in wells simultaneously coated with TDM and SL, indicating that SL suppresses TDM-induced TNF-alpha release from macrophages. Our results suggest a novel mechanism by which SL could contribute to virulence at early stage of mycobacterial infection or stimulation with the glycolipids by counteracting the immunopotentiating effect of TDM.

Differences in serological responses to specific glycopeptidolipid-core and common lipid antigens in patients with pulmonary disease due to Mycobacterium tuberculosis and Mycobacterium avium complex.

Disease due to the Mycobacterium avium complex (MAC) is one of the most important opportunistic pulmonary infections. Since the clinical features of MAC pulmonary disease and tuberculosis (TB) resemble each other, and the former is often difficult to treat with chemotherapy, early differential diagnosis is desirable. The humoral immune responses to both diseases were compared by a unique multiple-antigen ELISA using mycobacterial species-common and species-specific lipid antigens, including glycopeptidolipid (GPL)-core. The results were assessed for two patient groups hospitalized and diagnosed clinically as having TB or MAC pulmonary disease. Diverse IgG antibody responsiveness was demonstrated against five lipid antigens: (1) monoacyl phosphatidylinositol dimannoside (Ac-PIM2), (2) cord factor (trehalose 6,6'-dimycolate) (TDM-T) and (3) trehalose monomycolate from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) (TMM-T), and (4) trehalose monomycolate (TMM-M) and (5) GPL-core from MAC. Anti-GPL-core IgG antibody was critical, and detected only in the primary and the secondary MAC diseases with high positivity, up to 88.4 %. However, IgG antibodies against Ac-PIM2, TDM-T and TMM-T were elevated in both TB and MAC patients. Anti-TMM-M IgG antibody was also elevated in MAC disease preferentially, with a positive rate of 89.9 %, and therefore, it was also useful for the diagnosis of the disease. IgG antibody levels were increased at the early stages of the disease and declined in parallel to the decrease of bacterial burden to near the normal healthy control level, when the anti-mycobacterial chemotherapy was completed successfully. Unexpectedly, about 25 % of hospitalized TB patient sera were anti-GPL-core IgG antibody positive, although the specificity of GPL-core was sufficiently high (95.8 % negative in healthy controls), suggesting that a considerable number of cases of latent co-infection with MAC may exist in TB patients. Taken together, the combination of multiple-antigen ELISA using mycobacterial lipids, including GPL-core and TMM-M, gives good discrimination between healthy controls and sera from patients with TB or MAC disease, although for accurate diagnosis of TB more specific antigen(s) are needed.

Effect of inoculum conditioning on hydrogen fermentation and pH effect on bacterial community relevant to hydrogen production.

The effect of conditioning for a variety of inoculums on fermentative hydrogen production was investigated. In addition, the effects of pH condition on hydrogen fermentation and bacterial community were investigated. The effect of conditioning on hydrogen production was different depending on the inoculum types. An appreciable hydrogen production was shown with anaerobic digested sludge and lake sediment without conditioning, however, no hydrogen was produced when refuse compost and kiwi grove soil were used as inoculums without conditioning. The highest hydrogen production was obtained with heat-conditioned anaerobic digested sludge, almost the same production was also obtained with unconditioned digested sludge. The pH condition considerably affected hydrogen fermentation, hydrogen gas was efficiently produced with unconditioned anaerobic sludge when the pH was controlled at 6.0 throughout the culture period and not when only the initial pH was adjusted to 6.0 and 7.0. Hydrogen production decreased when the culture pH was only adjusted at the beginning of each batch in continuous batch culture, and additionally, bacterial community varied with the change in hydrogen production. It was suggested that Clostridium and Coprothermobacter species played important role in hydrogen fermentation, and Lactobacillus species had an adverse effect on hydrogen production.

Intact molecular characterization of cord factor (trehalose 6,6'-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry.

Cord factor (trehalose 6,6'-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 microg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [M+Na]+. Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H37Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with alpha-, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of alpha-, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens, the mass ion distribution due to polar mycolates was shifted lower than that from the Mycobacterium avium-intracellulare group. Since the physico-chemical properties and antigenic structure of mycolic acid of TDM affect the host immune responses profoundly, the molecular characterization of TDM by MALDI-TOF mass analysis may give very useful information on the relationship of glycolipid structure to its biological activity.

Clinical evaluation of serodiagnosis of active tuberculosis by multiple-antigen ELISA using lipids from Mycobacterium bovis BCG Tokyo 172.

The humoral immune responses of 69 active tuberculosis (TB) patients against three major mycobacterial lipid antigens, monoacyl phosphatidylinositol dimannoside (Ac-PIM2), trehalose 6,6'-dimycolate (TDM-T) and trehalose 6-monomycolate (TMM-T) from Mycobacterium bovis BCG Tokyo 172, were examined by ELISA. IgG antibodies from active TB patients were reactive against each of the three lipid antigens (Ac-PIM2, TDM-T and TMM-T), giving positive results of 42.0-59.4%. If tests were combined and an overall positive was scored when any of the three tests was positive, sensitivity was 71.0%, showing better discrimination between patients and normal subjects. Although this value is not satisfactory for the clinical diagnosis of active TB, it is still higher than values for Determinar TBGL (56.5%) and MycoDot (31.9%) test results, both of which are commercially available. IgG antibody responses to particular lipid antigens in an individual patient differed diversely between patients. Positive IgG antibody rates and IgG antibody levels to lipid antigens were mostly paralleled by the amount of mycobacteria excreted and by the severity of pathological lesions (size and cavity formation). Serologically positive responsiveness was shown in 60.0% of tuberculin skin test (TST)-negative TB patients. Furthermore, seropositivity for multiple-antigen ELISA in active TB patients was demonstrated in other possible immune-suppressed cases, such as senile, diabetes mellitus and fulminant TB patients. Therefore, in contrast to tests based on cellular immune responses such as the TST, the humoral immune responses of TB patients against mycobacterial lipid antigens were disease-specific and were shown to be useful for the early diagnosis of active TB disease in conjunction with smear and cultivation tests, even if cellular immune responses are decreased.