Perspectives for personalized therapy for patients with multidrug-resistant tuberculosis.

According to the World Health Organization (WHO), tuberculosis is the leading cause of death attributed to a single microbial pathogen worldwide. In addition to the large number of patients affected by tuberculosis, the emergence of Mycobacterium tuberculosis drug-resistance is complicating tuberculosis control in many high-burden countries. During the past 5 years, the global number of patients identified with multidrug-resistant tuberculosis (MDR-TB), defined as bacillary resistance at least against rifampicin and isoniazid, the two most active drugs in a treatment regimen, has increased by more than 20% annually. Today we experience a historical peak in the number of patients affected by MDR-TB. The management of MDR-TB is characterized by delayed diagnosis, uncertainty of the extent of bacillary drug-resistance, imprecise standardized drug regimens and dosages, very long duration of therapy and high frequency of adverse events which all translate into a poor prognosis for many of the affected patients. Major scientific and technological advances in recent years provide new perspectives through treatment regimens tailor-made to individual needs. Where available, such personalized treatment has major implications on the treatment outcomes of patients with MDR-TB. The challenge now is to bring these adances to those patients that need them most.

The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis.

We report that the spirochete B. burgdorferi induces progressive polyarthritis and carditis in mice with severe combined immunodeficiency syndrome (scid) but not in normal C.B-17 mice. The onset and severity of the disease were dependent on (a) the viability; (b) the infectivity; and (c) the dose of inoculated B. burgdorferi organisms. Infective spirochetes were isolated from both blood and joints of inoculated scid mice. These findings suggest that B. burgdorferi-induced chronic arthritis and carditis in mice develops independently of lymphocyte function and makes the scid mouse an attractive laboratory model to study the role of the immune system in experimental Lyme Borreliosis.

Author Correction: Enhanced tenacity of mycobacterial aerosols from necrotic neutrophils.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enhanced tenacity of mycobacterial aerosols from necrotic neutrophils.

The tuberculosis agent Mycobacterium tuberculosis is primarily transmitted through air, but little is known about the tenacity of mycobacterium-containing aerosols derived from either suspensions or infected neutrophils. Analysis of mycobacterial aerosol particles generated from bacterial suspensions revealed an average aerodynamic diameter and mass density that may allow distant airborne transmission. The volume and mass of mycobacterial aerosol particles increased with elevated relative humidity. To more closely mimic aerosol formation that occurs in active TB patients, aerosols from mycobacterium-infected neutrophils were analysed. Mycobacterium-infected intact neutrophils showed a smaller particle size distribution and lower viability than free mycobacteria. In contrast, mycobacterium-infected necrotic neutrophils, predominant in M. tuberculosis infection, revealed particle sizes and viability rates similar to those found for free mycobacteria, but in addition, larger aggregates of viable mycobacteria were observed. Therefore, mycobacteria are shielded from environmental stresses in multibacillary aggregates generated from necrotic neutrophils, which allows improved tenacity but emphasizes short distance transmission between close contacts.

In vivo virulence of Mycobacterium tuberculosis depends on a single homologue of the LytR-CpsA-Psr proteins.

LytR-cpsA-Psr (LCP) domain containing proteins fulfil important functions in bacterial cell wall synthesis. In Mycobacterium tuberculosis complex (Mtbc) strains, the causative agents of tuberculosis (TB), the genes Rv3484 and Rv3267 encode for LCP proteins which are putatively involved in arabinogalactan transfer to peptidoglycan. To evaluate the significance of Rv3484 for Mtbc virulence, we generated a deletion mutant in the Mtbc strain H37Rv and studied its survival in mice upon aerosol infection. The deletion mutant failed to establish infection demonstrating that Rv3484 is essential for growth in mice. Following an initial phase of marginal replication in the lungs until day 21, the Rv3484 deletion mutant was almost eliminated by day 180 post-infectionem. Interestingly, the mutant also showed higher levels of resistance to meropenem/clavulanate and lysozyme, both targeting peptidoglycan structure. We conclude that Rv3484 is essential for Mtbc virulence in vivo where its loss of function cannot be compensated by Rv3267.

Cellular immune reactivity to recombinant OspA and flagellin from Borrelia burgdorferi in patients with Lyme borreliosis. Complexity of humoral and cellular immune responses.

Patients with Lyme borreliosis (LB) usually develop a vigorous T cell response against the causative pathogen Borrelia burgdorferi, but little is known about the antigens recognized in the cellular response. Therefore, T cell reactivities against whole bacteria, recombinant 31-kD (outer surface protein A, [OspA]), and 41-kD proteins (flagellin) from B. burgdorferi were studied in patients with LB, non-LB patients, and healthy donors. In parallel, specific antibodies were determined by Western blot analysis. Virtually all patients with LB exhibited marked cellular responses to whole B. burgdorferi, which were significantly elevated compared with the control groups in both early and late disease stages. However, analyses using the purified antigens OspA and flagellin revealed considerable heterogeneity in the cellular reactivities among individuals as well as variations during the course of infection. T cell responses to OspA were significantly increased in patients with early LB compared with both control groups whereas in late-stage disease responses only exceeded those of non-LB patients and were not different from normal donors. Cellular immune reactivities to flagellin were significantly higher only in early LB compared with both control groups. Reciprocally, several control subjects demonstrated marked cellular responses to OspA and flagellin, suggesting that reactions to these proteins may not always be related to LB. T cell reactivity did not correlate well with the presence of specific antibodies. Almost all seropositive patients in both early and late stage LB had serum antibodies against flagellin, but antibodies to OspA were detectable only in a subset of late LB sera. These data demonstrate the complexity of the humoral and the cellular immune responses to components of B. burgdorferi.

CD1 and CD1-restricted T cells in infections with intracellular bacteria.

Glycolipid-specific, CD1a-, b- and c-dependent cytotoxic T cells have recently been shown to be involved in the host response against tuberculosis. These CD1 molecules 'sample' mycobacterial glycolipids from different intracellular sites in the infected cell. Additionally, upon microbial encounter, CD1d-dependent natural killer T cells promptly produce cytokines and perform regulatory activities. Here, we discuss the intracellular localization of CD1 molecules and mycobacterial lipids and the role of CD1-mediated T-cell responses in mycobacterial infections.

Killing of Borrelia burgdorferi by macrophages is dependent on oxygen radicals and nitric oxide and can be enhanced by antibodies to outer surface proteins of the spirochete.

Interaction of B. burgdorferi organisms with mouse bone marrow-derived macrophages (BMM phi) leads to phagocytosis of microorganisms, induction of nitric oxide (NO) and superoxide radicals (O2-) by BMM phi and killing of spirochetes. Destruction of spirochetes by BMM phi was quantified by a new method based on the release of radioactivity from spirochetes pre-labelled with [3H]adenine. Uptake of B. burgdorferi by BMM phi, which mainly occurs by coiling phagocytosis, generation of NO and O2- radicals as well as killing of spirochetes were significantly enhanced by pre-opsonization of spirochetes with monoclonal antibodies (mAb) to the outer surface proteins A and B but not with those to the periplasmic flagellin. Addition of inhibitors specific for NO and O2- radical synthesis either separately or together to cultures of BMM phi and spirochetes resulted in only partial reduction of the killing potential of effector cells. The data indicate that NO and O2- radicals are necessary, but not sufficient, for complete elimination of B. burgdorferi by macrophages. Together with previous findings that protection against B. burgdorferi infection is conveyed by humoral immune responses the present data indicate that one of the important functions of specific antibodies is their participation in macrophage-mediated control of spirochetes.

Distinct patterns of protective antibodies are generated against Borrelia burgdorferi in mice experimentally inoculated with high and low doses of antigen.

We have studied the development of clinical arthritis and the generation of protective antibodies in two normal, inbred strains of mice either infected by ticks or experimentally (subcutaneous) inoculated with increasing numbers of Borrelia burgdorferi organisms. AKR/N mice developed only mild and DBA/2 mice only marginal clinical arthritis irrespective of the route of infection or the numbers of spirochetes (10-10(8)) inoculated. In contrast, immunodeficient SCID mice developed severe chronic arthritis under similar conditions, but with a delayed onset at lower numbers of needle-inoculated spirochetes or after tick bite. AKR/N and DBA/2 mice inoculated with either 10(4) (and fewer) B. burgdorferi organisms or via experimentally infected ticks generated antibodies with specificities for a variety of B. burgdorferi antigens except those to the outer surface proteins A and B (OspA, OspB). In contrast, mice inoculated with more than 10(4) spirochetes (10(5)-10(8)) developed in addition antibodies to OspA and OspB. Most notably, all three types of immune sera taken from DBA/2 mice showed similar capacities to confer protection on SCID mice against subsequent challenge with viable B. burgdorferi organisms. The data not only demonstrate that the quality of humoral immune responses to B. burgdorferi in mice is determined by the antigenic load, they also indicate the existence of further protective antibodies with specificities distinct from OspA and OspB.

The outer surface lipoprotein A of Borrelia burgdorferi provides direct and indirect augmenting/co-stimulatory signals for the activation of CD4+ and CD8+ T cells.

Naive CD4+ and CD8+ T cells require two distinct signals to proliferate and to express effector functions [1]. One is provided by the antigen receptor on the T cell (TCR) after its encounter with antigenic peptides associated with class I or II major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC) [2]. The second signal, which is not antigen-specific but essential for proliferation and differentiation of naive T cells, is provided by co-stimulatory structures. The major co-stimulatory molecules for CD4+ T cells seem to be B7 [3], B7.2 [4,5], and heat-stable antigen (HSA) [6]. These molecules are expressed on a variety of naive and/or activated APC and bind to CD28 and CTLA-4 and possibly other, as yet undefined, TCRs [3,7]. Optimal T cell activation only occurs when co-stimulatory molecules and ligands for the TCR are expressed on the same APC [8,9]. However, co-stimulation for T cells may also be provided via bystander cells [8,9] or by glycoproteins of the extracellular matrix, like fibronectin [10] and laminin [11]. In this case, T-cell VLA integrins function as signaling molecules [10,11]. This indicates that antigen-specific T-cell activation may also occur in areas where antigens are presented in association with extracellular matrix proteins. The recent finding that the invasion protein of Yersinia spp. delivers co-stimulatory signals to anti-CD3-activated human T cells, most probably through the b1 integrins, suggests that bacterial products can also bind to contribute to the activation of T cells [12].(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Borrelia burgdorferi associated antigens by monoclonal antibodies.

In this paper, we present a series of murine mAb recognizing B. burgdorferi antigens. The antibodies were characterized by immuno-blotting and immuno-fluorescence studies using isolates of B. burgdorferi from North America and Europe, respectively. Moreover, reactivity of the antibodies with recombinant B. burgdorferi flagellin and OspA was studied. The results suggest these anti-B. burgdorferi mAb as valuable tools for the serological analysis of B. burgdorferi isolates and for affinity-purification of the respective proteins. Moreover, these mAb appear suitable to classify antigenic variants of B. burgdorferi and to study the protective capacity of antibodies in a murine model for B. burgdorferi infection.

Isolation of RNA from mycobacteria grown under in vitro and in vivo conditions.

Isolation of RNA from mycobacteria is very difficult to perform, and the yields are generally very low. We describe an approach to isolate RNA from mycobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker with the ease and efficiency of subsequent RNA purification on spin columns with silica gel-based membranes. This method is rapid, easy to perform and yields high amounts of pure, intact total RNA. Due to its safety, this method is applicable even to group 3 biological hazard organisms like Mycobacterium tuberculosis. By combining a method for the isolation of phagosomal bacteria from infected primary macrophages with the novel RNA isolation technique, we are able to monitor gene expression during infection even in bacteria which are rather resistant to genetic manipulation, like Mycobacterium bovis.

Infestation of rodents with larval Ixodes ricinus (Acari: Ixodidae) is an important factor in the transmission cycle of Borrelia burgdorferi s.l. in German woodlands.

The ecology of Borrelia burgdorferi Johnson et al. s.l. was investigated from 1987 to 1993 in a preserved woodland in western Germany near Bonn. In selected biotopes, host-seeking Ixodes ricinus L. were regularly collected by blanket dragging in 1987, 1988, and 1989 and screened for infection with B. burgdorferi. Rodents were trapped monthly between April and October in 1988, 1990, 1991, and in the winter of 1992-1993, examined for antibodies to B. burgdorferi s.l., and inspected for feeding ticks. Ticks collected from rodents were screened for spirochete infection. High numbers of host-seeking nymphs were consistently collected within a biotope characterized by humid and acid soils. The mean number of ticks was significantly lower in biotopes with permeable soils. All small mammals captured belonged to the species Apodemus flavicollis Melchior, A. sylvaticus L., and Clethrionomys glareolus Schreber. Of 11,680 ticks obtained from rodents, 11,674 were I. ricinus, with 97.9% of the ticks being larvae, 2.0% nymphs, and 0.1% females. Mean numbers of feeding ticks ranged from 3.4 to 117 larvae per rodent and from 0.0 to 0.64 nymph per rodent, respectively. High levels of larval infestation on rodents were recorded in the same biotope where high numbers of host-seeking nymphs were present. Members of the genus Apodemus were more heavily infested with I. ricinus larvae than C. glareolus. The mean infection prevalence in host-seeking ticks was found to be 1% for larvae, 5% for nymphs, and 10-20% for adults. The infection prevalence in host-seeking nymphs ranged from 1.1 to 15.4% according to the particular biotope. The values for specific infectivity for the Apodemus populations were positively correlated with the mean larval infestation, but not with nymphal infestation. The respective estimates for C. glareolus were much higher than those for Apodemus spp. in biotopes with low tick densities. However, specific infectivity of C. glareolus was substantially reduced at sites with high tick abundances. In biotopes with high numbers of infected I. ricinus, significantly more rodents were found to have antibodies to B. burgdorferi than in biotopes with low abundances of ticks. The data show that C. glareolus plays a different role as reservoir host species compared with the 2 Apodemus species. This and previous studies suggest that the degree of infestation with larval I. ricinus differentially modulates infectivity of host species for ticks. We conclude that immune processes in natural reservoir hosts induced by B. burgdorferi or I. ricinus bites (or both) are important regulatory factors in the transmission cycle(s) of B. burgdorferi.

No life without death--apoptosis as prerequisite for T cell activation.

The orchestrated death of infected cells is key to our understanding of CD8 T cell activation against pathogens. Most intracellular bacteria including Mycobacterium tuberculosis, the etiologic agent of tuberculosis, remain enclosed in phagosomes of infected macrophages. CD8 T cells play a critical role in defense of infection and recognize antigens originating from the cytosol presented by MHC-I molecules. Since mycobacteria do not gain access to the cytosolic MHC-I presentation pathway, the fundamental question as to how CD8 T cells encounter mycobacterial antigens remains to be solved. In this review, we focus on solutions for this enigma and describe the detour pathway of T cell activation. Mycobacteria induce cell death of infected macrophages which thereby leave a last message by releasing apoptotic vesicles. Subsequently, these antigen-containing entities are engulfed by dendritic cells which process the mycobacterial cargo for efficient antigen presentation and CD8 T cell activation. Since the dying infected cell is the origin of a protective T cell response destined to preserve life and individuality, the detour pathway represents an altruistic principle at a cellular level which corresponds to the macroscopic world where death is the precondition to perpetuate the living.

CD1 molecules and CD1-dependent T cells in bacterial infections: a link from innate to acquired immunity?

The MHC class I-like, non-polymorphic CD1 molecules represent a novel system for the presentation of glycolipid antigens to T lymphocytes. CD1-mediated T cell responses appear to play distinct roles during bacterial infections such as in tuberculosis. This review deals with two aspects of CD1-mediated immune reactions. First we discuss the role of group II CD1-dependent NK T cells in bacterial infection. Second, we provide an insight into differential intracellular meeting points for antigen processing between group I CD1 molecules, mycobacteria and mycobacterial glycolipid antigens.

Mode of inoculation of the Lyme disease agent Borrelia burgdorferi influences infection and immune responses in inbred strains of mice.

Mice were infected with Borrelia burgdorferi by infection via Ixodes ricinus and experimental inoculation to determine whether transmission rates of spirochetes and antibody responses are influenced. Mice infected by the natural route were substantially more infective for ticks; two- to sixfold more tick larvae were positive for B. burgdorferi than those fed on experimentally inoculated mice. In natural infection, spirochetemia may be greater or spirochetes may be more accessible for transmission. Thus, this form of xenodiagnosis could be used to determine levels of spirochetes in the vertebrate host. Similar levels of antibody were present in all mice; however, those infected by the natural route lacked antibodies to outer surface proteins (Osp) A and B. The small antigen dose given through a tick bite may not have been sufficient to induce rapid OspA or OspB antibodies, thereby allowing the later development of higher levels of spirochetemia.