The unique tropism of Mycobacterium leprae to the nasal epithelial cells can be explained by the mammalian cell entry protein 1A.

Leprosy is a chronic infection where the skin and peripheral nervous system is invaded by Mycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet for M. leprae. Mce1A protein (442 aa) is coded by mce1A (1326 bp) of M. leprae. The Mce1A homolog in Mycobacterium tuberculosis is known to be associated with M. tuberculosis epithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that Mce1A of M. leprae is also associated with epithelial cell entry. This study is aimed at identifying particular sequences within Mce1A associated with M. leprae epithelial cell entry. Recombinant proteins having N-terminus and C-terminus truncations of the Mce1A region of M. leprae were created in Escherichia coli. Entry activity of latex beads, coated with these truncated proteins (r-lep37 kDa and r-lep27 kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316-921 bp region was divided into three sub-regions: 316-531 bp (InvX), 532-753 bp (InvY), and 754-921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface of E. coli. Entry of these E. coli into monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. Only E. coli harboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa-InvXd, containing sequences 1-24 aa, 25-46 aa, 47-57 aa, and 58-72 aa, respectively. Recombinant E. coli, expressing each of InvXa-InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of Mce1A are important for M. leprae invasion into nasal epithelial cells.

Study of the potential of the air lift bioreactor for xylitol production in fed-batch cultures by Debaryomyces hansenii immobilized in alginate beads.

Cell immobilization has shown to be especially adequate for xylitol production. This work studies the suitability of the air lift bioreactor for xylitol production by Debaryomyces hansenii immobilized in Ca-alginate operating in fed-batch cultures to avoid substrate inhibition. The results showed that the air lift bioreactor is an adequate system since the minimum air flow required for fluidization was even lower than that leading to the microaerobic conditions that trigger xylitol accumulation by this yeast, also maintaining the integrity of the alginate beads and the viability of the immobilized cells until 3 months of reuses. Maximum productivities and yields of 0.43 g/l/h and 0.71 g/g were achieved with a xylose concentration of 60 g/l after each feeding. The xylose feeding rate, the air flow, and the biomass concentration at the beginning of the fed-batch operation have shown to be critical parameters for achieving high productivities and yields. Although a maximum xylitol production of 139 g/l was obtained, product inhibition was evidenced in batch experiments, which allowed estimating at 200 and 275 g/l the IC50 for xylitol productivity and yield, respectively. The remarkable production of glycerol in the absence of glucose was noticeable, which could not only be attributed to the osmoregulatory function of this polyol in conditions of high osmotic pressure caused by high xylitol concentrations but also to the role of the glycerol synthesis pathway in the regeneration of NAD(+) in conditions of suboptimal microaeration caused by insufficient aeration or high oxygen demand when high biomass concentrations were achieved.

Application of Hansen solubility parameters for understanding and prediction of drug distribution in microspheres.

In an emulsion solvent extraction/evaporation process for the preparation of microspheres the employed solvents have a tremendous influence on the characteristics of the resulting particles. Nevertheless the solvent selection is often based on empirical data rather than on calculated values. The purpose of this investigation was to use the concept of solubility parameters for interpretation and improved understanding of solvent effects in the process of microparticle preparation. Partial solubility parameters of 3-{2-[4-(6-Fluor-1,2-benzisoxazol-3-yl)piperidino]ethyl}-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-on, which was used as a model drug, were determined experimentally using an extended Hansen regression model. Poly(lactide-co-glycolide) microparticles were prepared with an emulsion solvent removal process employing methylene chloride and its mixtures with benzyl alcohol and n-butanol. It could be shown, that the encapsulation efficiency was influenced by the change of the solvent composition during the extraction process. Furthermore the solvent selection had an essential influence on the morphological state of the drug and it could be shown and explained, that by a decrease of the dissolving power a completely amorphous product was obtained.

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection.

Of the hundreds of new tuberculosis (TB) vaccine candidates, some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65+CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Engulfed pathogen-induced apoptosis in haemocytes of giant freshwater prawn, Macrobrachium rosenbergii.

Haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, were investigated for the induction of apoptosis after phagocytosis of pathogenic yeasts, bacteria and non-pathogenic latex beads in vitro. Isolated haemocytes of M. rosenbergii were cultured at a ratio of 1:50 haemocytes to pathogen with the yeast Debaryomyces hansenii, the bacteria Aeromonas hydrophila or Enterococcus faecium, or with latex beads at 25 degrees C for 2 h, followed by washing to remove free particles. At least 200 haemocytes were counted to determine the phagocytosis rate, and the results showed that haemocytes engulfed latex beads at a higher rate than the aquatic pathogens. By transmission electron microscopy, the yeast- or bacterium-engulfing haemocytes displayed morphological changes characteristic of apoptosis, including formation of cytoplasmic vacuoles, chromatin condensation and fragmentation of nuclei. This pathogen-induced apoptosis was further confirmed by DNA laddering and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end-labelling) assays. Neither haemocytes treated with latex beads nor uninfected haemocytes (control group) showed signs of apoptosis after 48 h in culture.

Anti-mycobacterial immunity induced by a single injection of M. leprae Hsp65-encoding plasmid DNA in biodegradable microparticles.

A single sub-cutaneous injection of a plasmid DNA encoding a mycobacterial heat shock protein 65 (Hsp65) entrapped in biodegradable poly(lactic-co-glycolic acid) microspheres produced high titers of antibodies, measured 5 months after the injection in BALB/c mice. Splenocytes secreted IFN-gamma and exerted an anti-bacterial effect on macrophages infected in vitro with Mycobacterium tuberculosis. The results are encouraging with regard to obtaining good compliance and vaccination coverage with candidate plasmid DNA vaccines, especially in developing countries.

Release mechanism of insulin encapsulated in trehalose ester derivative microparticles delivered via inhalation.

The aim of this study was to evaluate properties of amorphous oligosaccharide ester derivative (OED) microparticles in order to determine drug release mechanisms in the lung. Trehalose OEDs with a wide range of properties were synthesised using conventional methods. The interaction of spray dried amorphous microparticles (2-3 microm) with water was investigated using attenuated total reflectance Fourier transform infra-red spectroscopy (ATR-FTIR) and dynamic vapour sorption (DVS). The in vivo performance of insulin/OED microparticles was assessed using a modified Higuchi kinetic model. A modified Hansen solvent parameter approach was used to analyse the interactions with water and in vivo trends. In water or high humidity, OED powders absorb water, lose relaxation energy and crystallise. The delay of the onset of crystallisation depends on the OED and the amount of water present. Crystallisation follows first order Arrhenius kinetics and release of insulin from OED microparticles closely matches the degree of crystallisation. The induction period depends on dispersive interactions between the OED and water while crystallisation is governed by polarity and hydrogen bonding. Drug release from OED microparticles is, therefore, controlled by crystallisation of the matrix on contact with water. The pulmonary environment was found to resemble one of high humidity rather than a liquid medium.

Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae.

The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.

Interactions between cationic liposomes and an antigenic protein: the physical chemistry of the immunoadjuvant action.

The 18 kDa antigenic protein from Mycobacterium leprae (P) or its N-acyl derivative (AP) was incorporated in dioctadecyldimethylammonium bromide (DODAB) liposomes in water or in phosphate-buffered saline (PBS). In water, 100% P incorporation in liposomes contrasts with 65% in PBS. There is 75-80% AP incorporation to liposomes in water against 55-65% in PBS, showing that attachment of hydrophobic residues to the protein, instead of increasing, further decreases incorporation to the liposomes. From protein adsorption on latex, P affinity is larger than AP affinity for the latex surface whereas limiting adsorption for AP is much larger than that obtained for P, possibly due to AP aggregation in solution. P-induced rupture of liposomes containing [14C]sucrose was evaluated from dialysis of protein/liposomes mixtures. In water, P incorporation to the liposomes causes leakage of radioactive contents contrasting with the absence of leakage for P incorporation in PBS. Immunization tests for delayed type hypersensitivity indicate a enhancement of cell-mediated immunological response towards P/DODAB complexes that is not obtained for the isolated protein. Absence of leakage for P in PBS is associated with a P "lying-over" on the liposome and optimization of protein presentation to the immunological system.

Antigen-coated latex particles as a model system for probing monocyte responses in leprosy.

To study responses to Mycobacterium leprae antigens, we developed an in vitro model system in which latex particles coated with M. leprae sonic extract (MLSON) antigen were presented to monocytes. Uptake and oxidative response as measured by superoxide production to these antigens were investigated. Phagocytosis of MLSON-coated particles was greater than that of control particles in monocytes from both leprosy patients and controls from leprosy-endemic areas; uptake of MLSON-coated particles was higher in monocytes from lepromatous leprosy patients than in cells from tuberculoid leprosy patients and controls. In both patients and controls, uptake of latex particles coated with leprosy antigens triggered very little reduction of nitroblue tetrazolium although the cells were capable of mounting a respiratory burst. Antigen-coated latex particles can therefore be used as a tool to investigate monocyte responses to M. leprae and individual recombinant antigens.

Microtiter particle agglutination test for diagnosis of leprosy.

The results of studying the microtiter particle agglutination (MPA) test for detecting anti-Mycobacterium leprae antibodies in blood sera are presented. The serodiagnostic test is based on the agglutination of colored polyacrolein latex microparticles (PAMP) conjugated with 3,6-di-O-methyl-D-glucose (DMG). Sera from 45 leprosy patients (LL, BL), 34 leprosy contacts, and 148 control subjects were investigated by the MPA test. A correlation between the anti-M. leprae antibodies and the bacterial load was found. In many long-treated leprosy patients increased titers of anti-DMG antibodies were observed, which might be due to specific polyneuritis in them. Four contacts of leprosy patients were also positive in the MPA test. "Nonleprosy" sera did not react in the test. The method proposed proved to be of high specificity and sensitivity for the serological diagnosis of leprosy. The rapidity, simplicity, and visual assessment of the results allow the method to be used in the field for epidemiological studies of leprosy contacts and the general population in leprosy-endemic areas.

Field utility of phenolic glycolipid coated latex agglutination test for rapid detection of bacilliferous leprosy cases.

Serum samples were collected from eighty-three leprosy patients and twenty-five healthy controls supposedly not exposed to Mycobacterium leprae infection. Phenolic glycolipid-1 coated latex agglutination test (PGL-LAT) was carried out with the serum samples to detect antibodies specific to M. leprae. Samples showing positive agglutination were 50% in the lepromatous leprosy (LL) group showing no erythema nodosum leprosum (ENL) complications, 66.6% in LL group with ENL complication, 60% in borderline lepromatous (BL) group, 50% in borderline (BB) and 33.3% in borderline tuberculoid (BT). The patients belonging to the tuberculoid (TT) group and most of the long-term treated patients were interestingly negative, and so were sera from all the healthy controls. PGL-LAT developed by us therefore is specific and a fairly sensitive technique to detect antibodies specific to M. leprae and will be very useful in field conditions.

Respiratory burst metabolic status of macrophages in experimental leprosy.

Peritoneal macrophages from uninfected controls and Mycobacterium leprae infected Swiss albino mice were studied for their respiratory burst (RB) activity at different time intervals. The RB metabolic activity of macrophages declined significantly after 3 month infection using latex (p less than 0.001) and M. leprae (p less than 0.01) as stimuli. However, significant rise (p less than 0.001) in the oxidative metabolic activity was seen at 6 and 9 months postinfection period on stimulation with both the stimuli. The sharp rise in the oxidative metabolic status at peak period of infection in the experimental animals suggests that the macrophages are functionally normal though M. leprae is unable to trigger the respiratory burst sufficiently.

A rapid latex agglutination test for detection of antibodies in tuberculosis and Hansen's disease.

Antigens of Mycobacterium w, a saprophytic fast growing organism having antigenic epitopes cross-reactive with Mycobacterium leprae and Mycobacterium tuberculosis, were coated on to latex beads (0.33 micron Zn size), and the reactivity tested with sera of tuberculosis and Hansen's disease (HD) patients. Seventy nine percent of lepromatous leprosy (LL) and eighty five percent of pulmonary tuberculosis (TB) patients sera showed an agglutination reaction easily read by naked eye. Specificity of the test was further checked by testing sera of non-mycobacterial infection cases and all of them were found negative. Among apparently healthy controls, 4.3% were found positive from non-endemic and 8.8% from endemic area. The sensitivity of the assay is further enhanced from 78.7% to 90.4% and 85.7% to 91.6% in both LL HD and pulmonary tuberculosis respectively, by using immune complexes extracted from the patients sera. Potential of these antigen coated beads to detect the two major human mycobacterial disease, LL HD and pulmonary TB was also put evidence in a double blind study on coded sera samples obtained from various hospitals in India. The antigen coated beads are stable for upto 6 months at 4 degrees C. The latex slide agglutination test reported here, is simple, rapid, easy to perform and can be used even in rural areas of developing countries.

Phagocytosis in leprosy. 2. Production of superoxide by circulating blood leukocytes from lepromatous patients.

The ability of polymorphonuclear leukocytes from lepromatous patients to produce superoxide (O-2) anion while phagocytizing latex particles was studied. The results were compared with those obtained in a group composed of normal individuals. No differences were found between lepromatous and normal groups. No differences were found either when the comparison was made between patients showing any form of leprosy reaction and patients without leprosy complications at the time of the study. Together with other results previously reported, our findings suggest that PMN from lepromatous patients are not different from PMN from healthy individuals with respect to their ability to generate and show the metabolic oxidative changes (as measured by the NBT- and the O-2-tests) induced by in vitro phagocytosis of latex particles. These metabolic activities remain normal even when PMN may show alterations at other levels of the phagocytic process.