Fibroblasts as host cells in latent leishmaniosis.

Intracellular parasites are known to persist lifelong in mammalian hosts after the clinical cure of the disease, but the mechanisms of persistence are poorly understood. Here, we show by confocal laser microscopy that in the draining lymph nodes of mice that had healed a cutaneous infection with Leishmania major, 40% of the persisting parasites were associated with fibroblasts forming the reticular meshwork of the lymph nodes. In vitro, both promastigotes and amastigotes of L. major infected primary skin or lymph node fibroblasts. Compared with macrophages, cytokine-activated fibroblasts had a reduced ability to express type 2 nitric oxide synthase and to kill intracellular L. major. These data identify fibroblasts as an important host cell for Leishmania during the chronic phase of infection and suggest that they might serve as safe targets for the parasites in clinically latent disease.

Apoptotic cell death in activated monocytes following incorporation of clodronate-liposomes.

The present study was performed to elucidate whether sterically stabilized liposomes laden with clodronate, which lead to depletion of macrophages (Mphis) and amelioration of experimental autoimmune arthritis in vivo, selectively affect cells of the mphi lineage in vitro. The rates of incorporation of drug-free, fluorescent liposomes and the rates of cell death following exposure to clodronate-liposomes were assessed in human peripheral blood monocytes, as well as in polymorphonuclear leukocytes (PMNs), T cells, endothelial cells, and fibroblasts, both at rest and following activation. Gel electrophoresis of nuclear extracts and ultrastructural analyses were performed to identify the modality of cell death. Monocytes, particularly upon activation, were more efficient in incorporating sterically stabilized liposomes than all other cells except PMNs. Twenty percent of resting monocytes and up to 65% of activated monocytes died within 24 h of exposure to clodronate-liposomes, whereas the other cell types, including PMNs, remained unaffected. Activated monocytes exposed to clodronate-liposomes, but not resting or activated monocytes exposed to drug-free liposomes, showed clear signs of apoptotic cell death. In most of the assays, sterically stabilized liposomes were more efficient than conventional phosphatidylcholine-liposomes. Sterically stabilized clodronate-liposomes preferentially affect cells of the mphi lineage, particularly if activated. Selective elimination of activated Mphis by apoptosis may explain both therapeutic efficacy and safety of clodronate-liposomes in experimental models of autoimmunity.

Phagocytosis of microorganisms by means of overshooting pseudopods: where do we stand?

During the endocytic uptake of particulate material such as microorganisms, the transition from the engulfment step to the internalization step of phagocytosis may be disturbed. Thus, the pseudopods flanking the particles do not close to a phagosome, but lie on top of each other. This uncoupling of pseudopod extension and phagosome formation provides useful information about the regular course of phagocytosis. Experimental models on the phenomena of coiling and overlapping phagocytosis have so far been established with legionellas, spirochetes, trypanosomatids, fungal cells, and zymosan.

Immunohistochemical study of extracellular material in the aged human synovial membrane.

Types III, IV, VI collagen and laminin distribution in synovial tissue of seven autopsy knee joints from old human donors (69-94 years of age) were investigated with immunocytochemical and immunohistochemical methods. The synovial intima is separated from the subintimal tissue by an intermediate fibrillar zone rich in staining for type III collagen. In the intima basement membrane-like material associated with synovial lining cells stains for type IV collagen and laminin. Fine fibrils surrounding the lining cells stain for type VI collagen. In two of the cases type VI collagen occurs mainly as long-spacing collagen, the distinct aggregated form of type VI collagen. This staining pattern was qualitatively the same in all different regions and cases investigated. However, considerable quantitative differences were seen.

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.

Phagocytes from both vertebrate and invertebrate species use "coiling" phagocytosis.

Coiling phagocytosis has been observed previously only by chance, and there has been no systematic investigation of this uptake mechanism. Therefore, a comparative electron microscopical study was performed. Different human and murine cell populations, phagocytes from various vertebrate and invertebrate species, and predatory amoebae were incubated with Borrelia burgdorferi, one of the microbes known to induce coiling phagocytosis, to study the uptake mechanisms used. In this model, coiling phagocytosis was observed with both vertebrate and invertebrate species but not with amoebae. With cells from humans and mice, this uptake mechanism was restricted to phagocytic cells of myeloid origin. The coiled membrane gaps did not give rise to phagosomes; instead, membrane fusion was followed by membrane dissipation. Thus, coiling of B. burgdorferi apparently is an alternative uptake mechanism used by metazoan phagocytes, involving special membrane processing. However, coiling phagocytosis may show different features with different microbes.

Hydroxyurea for treatment of unresectable and recurrent meningiomas. I. Inhibition of primary human meningioma cells in culture and in meningioma transplants by induction of the apoptotic pathway.

Meningiomas, which invade intracranial bone structures and the adjacent connective tissue, are frequently unresectable because of their aggressive and recalcitrant growth behavior. They have a high recurrence rate, and in approximately 10% of these tumors there is an increased risk of malignancy. Significant morbidity and mortality rates associated with recurrent meningiomas demand nonsurgical approaches. To date, adjuvant hormonal treatment has not proven beneficial. The anticancer drug hydroxyurea was therefore tested for its potential use in the treatment of meningiomas. Early-passaged cell cultures were established from 20 different meningiomas. The addition of 5 x 10(-4) and 10(-3) M hydroxyurea over a period of 5 to 9 days resulted in a remarkable decrease in cell proliferation and even blocked tumor cell growth when compared with untreated cells. A significant arrest of meningioma cell growth in the S phase of the cell cycle was revealed on DNA flow cytometry. Electron micrographs of hydroxyurea-treated tumor cells showed ultrastructural features consistent with apoptosis, and light microscopy demonstrated DNA fragmentation by in situ DNA strand break labeling. Short-term treatment of meningioma cell cultures with hydroxyurea for 24 to 48 hours resulted in discrete oligonucleosomal fragments (DNA ladder), another characteristic sign of apoptosis. In addition to the in vitro studies, tissue from five different meningiomas was transplanted into nude mice followed by treatment with 0.5 mg/g body weight hydroxyurea over 15 days. In situ DNA strand break labeling demonstrated DNA fragmentation in distinct regions with different tumor cell densities in all hydroxyurea-treated meningioma transplants. These data provide evidence that hydroxyurea is a powerful inhibitor of meningioma cell growth, most likely by causing apoptosis in the tumor cells. Thus, hydroxyurea may be a suitable chemotherapeutic agent for the long-term treatment of unresectable or semi- to malignant meningiomas, or for preventing recurrent growth of meningiomas after resection.

Hydroxyurea for treatment of unresectable and recurrent meningiomas. II. Decrease in the size of meningiomas in patients treated with hydroxyurea.

In this paper the authors present the first evidence that meningiomas respond to treatment with hydroxyurea. Hydroxyurea was administered as an adjunct chemotherapeutic treatment in patients with recurrent and unresectable meningiomas. Hydroxyurea was used because experimental data demonstrated that it inhibits growth of cultured human meningioma cells and meningioma transplants in nude mice by inducing apoptosis. The authors therefore treated four selected patients with hydroxyurea. All patients had undergone multiple gross resections and all except one received radiotherapy. Three patients with recurrent Grade I meningiomas assessed according to World Health Organization (WHO) guidelines received hydroxyurea because of an increased tumor growth rate, documented by magnetic resonance (MR) imaging, within a 6- or 12-month interval. A fourth patient with a malignant meningioma (WHO Grade III) began a course of treatment with hydroxyurea immediately after his sixth palliative operation without waiting for another relapse to be demonstrated on MR imaging. Because of their location and invasive growth behavior none of the meningiomas could have been removed completely by surgical intervention. All patients received hydroxyurea at a dosage level of 1000 to 1500 mg/day (approximately 20 mg/kg/day). In a man with a large sphenoid wing meningioma invading the right cavernous sinus and the temporal base, the intracranial tumor mass was reduced by 60% during 6 months of treatment. A woman with a large ball-shaped meningioma of the right sphenoid wing invading the cavernous sinus exhibited a 74% decrease of the initial tumor volume in 10 months of treatment with oral hydroxyurea. Serial MR images obtained monthly revealed that the process of size reduction was continuous and proportionate. The shrinkage of the tumor was accompanied by a complete remission of symptomatic trigeminal neuralgia after 2 months and by improved abducent paresis after 5 months. The third patient had a slowly growing meningioma that exhibited a 15% reduction in mass when reassessed after 5 months of hydroxyurea treatment. The fourth patient with the malignant meningioma in the left cerebellopontine angle has had no recurrence for 24 months. Long-term treatment with hydroxyurea may result in full remission of tumors in meningioma patients. The preliminary data indicate that hydroxyurea provides true medical treatment in patients with unresectable and recurrent meningiomas, replacing palliative surgery and radiotherapy in the management of this disease.

Studies on the pathogenesis and treatment of Lyme arthritis.

Lyme arthritis is one of the most common clinical manifestations of Lyme borreliosis. It is caused by an intraarticular infection with Borrelia (B.) burgdorferi. A small number of bacteria are liable to provoke severe arthritis by inducing mechanisms (including the induction of cytokines and chemokines) that amplify the inflammatory response. The cellular immune response against B. burgdorferi is characterised by a predominant T helper cell type 1 (Th1) pattern that appears to be inadequate to overcome the infection. In most cases, Lyme arthritis may be cured by antibiotic therapy. A brief summary of current recommendations for the treatment of Lyme arthritis in adults and children is given in this article. However, about 10% of Lyme arthritis patients do not respond sufficiently to antibiotic treatment. Two not mutually exclusive pathogenetic concepts of these treatment-resistant cases will be discussed in the present study: persistent infection and infection-induced immunopathology.

Leishmania-host-cell interaction: complexities and alternative views.

Leishmania are protozoan parasites that infect various mammalian species, including humans. It is generally thought that random attachment of the flagellated promastigotes to mononuclear phagocytes initiates their uptake via circumferential pseudopods. Intracellularly, the promastigotes become located in phagolysosomes in which they transform to and survive as 'aflagellated' amastigotes that hide their shortened flagellum within the flagellar pocket. Unrestricted replication of these amastigotes is assumed to cause the eventual burst of the host cell, thereby releasing the infectious parasites. Here, Mike Rittig and Christian Bogdan review a large body of literature containing potentially important but poorly appreciated findings, which together with recent results, argue for Leishmania-host-cell interactions that are much more complex than generally thought.

Innervation of the ciliary process vasculature and epithelium by nerve fibers containing catecholamines and neuropeptide Y.

The ciliary processes (CPs) in the rat were investigated for the presence of neuropeptide Y (NPY)-like immunoreactivity and for histofluorescence indicating catecholamine (CAM)-ergic innervation in the rat, rabbit and cynomolgus monkey. Special attention was paid to those CP vessel segments which in previous studies showed distinct reactions on application of CAM or NPY, and on the respective sections of the CP epithelium. In the rat and cynomolgus monkey most CAM-ergic nerve fibers concentrated along the terminal arterioles and the epithelium of the anteriormost portion of the major CPs. In comparison, the rabbit displayed most intense CAM-ergic innervation along the terminal arterioles and the epithelium of both the iridial processes and the anterior portion of the major CPs. The NPY-ergic nerve fibers built up a dense subepithelial nervous plexus in the anterior portion of the rat CPs, diminishing towards the posterior CPs. Also, many NPY-ergic fibers were found along the terminal arterioles of the anterior CPs. The findings demonstrate that CAM- and NPY-ergic nerve fibers preferentially supply vasculature and epithelium of the anterior ciliary processes, suggesting a crucial function of these structures for the precise regulation of aqueous humor formation.

Immunohistochemical localization of hyaluronan synthase in cornea and conjunctive of cynomolgus monkey.

Distribution of hyaluronan synthase was investigated in cornea and conjunctiva of Cynomolgus monkeys (Macaca fascicularis) using polyclonal antibodies against the streptococcal enzyme. Strong immunoreaction was found in the cell membranes of the corneal endothelium, corneal epithelium, and most of the conjunctival epithelium. In the corneal epithelium all cells except the basal ones stained. In the conjunctiva all cylindrical cells stained, whereas among the goblet cells one type showed intense membrane staining, the other remained unstained. In the limbal portion of the conjunctival epithelium, which in many other respects differs morphologically and functionally from the remaining conjunctiva, all membranes of the different layers of the stratified epithelium except the most superficial ones, appeared unstained. Staining was also seen in all stromal fibroblasts and capillary endothelial cells.

Hyaluronan synthase immunoreactivity in the anterior segment of the primate eye.

The anterior segment of human and cynomolgus monkey eyes was investigated for the presence of hyaluronan (HA) synthesizing cells using a polyclonal antibody against the enzyme HA synthase (HAS). In the chamber angle region the most intense staining was seen in the cell membranes of the corneal endothelium and in monkey eyes in the cells covering the posterior extension of the cornea (the operculum). The trabecular meshwork cells of the uveal and inner corneoscleral lamellae were also intensely stained. On the other hand, no staining was observed in the trabecular cells of the outer corneoscleral and the cribriform meshwork. The cell membranes of the inner wall endothelium of Schlemm's canal were labelled only at their luminal surface. In the iris stroma and the trabeculum ciliare (the ciliary body band), labelled cells were also found, whereas the connective tissue of the ciliary muscle and the muscle itself did not contain HAS-positive cells. In the ciliary processes immunoreactivity was seen in the non-pigmented epithelial cells (NPE) covering the anterior tips of the processes, suggesting that HA found in the aqueous humor is produced by these cells. The pars plana NPE showed the most intense staining in the cells directly adjacent to the ora serrata region. The hyalocytes found in the neighborhood of the pars plana also showed intense HAS immunoreactivity. It is likely that both hyalocytes and NPE cells of the posterior pars plana release HA into the vitreous.

Lectin-binding sites in the anterior segment of the human eye.

The anterior segment of the human eye was screened for differences in the lectin-binding patterns of Con A, PNA, GS-I, WGA, SBA, DBA, and UEA-I to enable cell typing for cell-culture purposes. An immunohistochemical technique combining an indirect antibody-lectin method with the avidin-biotin system was used. Con A and WGA were bound by all cells except the conjunctival goblet cells. UEA-I was exclusively bound by both vascular endothelial cells and some corneal and conjunctival epithelial cells. The binding of GS-I, PNA, SBA, and DBA showed an uneven pattern and differed among the cases investigated. The reasons for these differences are not clear. Our results indicate that the usefulness of lectins for cell-typing purposes is restricted and must be determined for every case.

[Electron microscopy and immunohistochemical studies of the intraocular pressure lowering effect of prostaglandin F2 alpha]. / Elektronenmikroskopische und immunhistochemische Untersuchungen zur augendrucksenkenden Wirkung von Prostaglandin F2 alpha.

Topically applied prostaglandin F2 alpha (PGF2 alpha) has been shown to lower intraocular pressure in cynomolgus monkeys. In this study the morphological changes following topical treatment of seven cynomolgus monkeys with 4 micrograms PGF2 alpha isopropylester for 5-8 days were investigated and compared with the eyes of five normal animals. Quantitative light microscopical, ultrastructural and immunhistochemical methods were used. No cellular signs of inflammation were seen in any of the eyes. Slight edema in the most anterior part of the ciliary processes occurred in most eyes, but only in parts of the circumference. The most pronounced change was dilation of the intramuscular spaces within the ciliary muscle. No changes were observed in the extracellular matrix within the muscle bundles, which consist partly of type IV and VI collagen, laminin and fibronectin. In the connective tissue between the muscle bundles, loss of collagen type I and III fibrils was observed. Additionally, macrophages were found with phagolysosomes containing phagocytized collagen fibrils. We suggest that loss of extracellular material leads to a widening of the uveoscleral outflow pathways of ciliary muscle and thereby to a reduction in intraocular pressure.

Biomonitoring of pJP4-carrying Pseudomonas chlororaphis with Trb protein-specific antisera.

The transfer of catabolic genes on conjugative plasmids to indigenous organisms from which they may spread further into the community allows the introduction of new biodegradative pathways for metabolic conversion of pollutants to the community. Biomonitoring of IncP plasmid pJP4-carrying Pseudomonas chlororaphis from the rhizosphere of Arabidopsis thaliana was achieved using antisera specific for proteins from the plasmid transfer machinery. Antisera were generated that recognized TrbC and TrbF, the putative major and minor components of pJP4-determined pili, respectively, and the putative lipoprotein TrbH. Cell fractionation studies showed association of TrbC, TrbF and TrbH with the cells and suggested that TrbC and TrbF are part of extracellular pJP4-determined pili. TrbF and TrbH antisera allowed specific detection of IncP compared with IncN or IncW plasmid-carrying cells and even permitted differentiation between bacteria carrying IncPalpha plasmid RP4 and IncPbeta plasmid pJP4. Immunofluorescence microscopy was applied to detect TrbF and TrbH signal at the cell periphery, allowing distinction from autofluorescing cells and soil debris. In situ experiments showed specific recognition of pJP4-carrying cells from laboratory cultures, as well as from the rhizosphere of A. thaliana grown in natural soil. After co-inoculation of donor P. chlororaphis pJP4 and recipient Ralstonia eutropha, a combination of immunofluorescence and oligonucleotide hybridization techniques permitted the detection of plasmid transfer between both organisms in the A. thaliana rhizosphere. This strategy may be generally applicable for the analysis of plasmid transfer in natural ecosystems.

An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme borreliosis.

The present study aimed at developing an optimized PCR protocol fro the sensitive and specific detection of all three Borrelia burgdorferi genospecies pathogenic to humans in Lyme borreliosis patients. A rapid DNA extraction method using alkaline lysis was introduced and was found to be superior to other DNA extraction methods. Nested PCR was performed with primer sets targeting the plasmid-located ospA gene and a chromosomal gene segment encoding a 66-kDa protein (p66). In spiked synovial fluid (SF) fewer than three borreliae/sample were detected. The specificities of the amplicons were confirmed by Southern blot analysis with PCR-derived probes. Urine, cerebrospinal fluid (CSF), and SF specimens from 57 patients with Lyme borreliosis and from 58 controls were examined. In clinical samples the diagnostic sensitivity of PCR was 85% with SF samples, 79% with urine samples, and 91% with paired SF-urine samples from patients with Lyme arthritis and was 79% with CSF samples, 45% with urine samples, and 87% with paired CSF-urine specimens from neuroborreliosis patients. One patient each with neuroborreliosis and with Lyme arthritis had PCR-positive urine samples only. In 17% of all cases both primer sets yielded positive results, while the other patients were positive with only one primer set. Among these, more positive results were obtained with the p66 gene primer than with the ospA primer. The specificity exceeded 99%. We conclude that DNA from B. burgdorferi sensu lato species can sensitively and specifically be detected with the optimized PCR method described. At least two different primer sets should be used, and whenever possible, urine and CSF or SF should be analyzed in parallel to achieve maximum sensitivity of the test. This protocol, therefore, considerably enhances the diagnostic power of PCR in patients with B. burgdorferi infection.

Human dendritic cells phagocytose and process Borrelia burgdorferi.

There is strong evidence that the immune response to Borrelia burgdorferi (Bb) contributes to the pathogenesis of Lyme disease. Bb are transmitted by ticks to the skin, which is particularly rich in dendritic cells (DC). The initial reaction of these APCs may already set the course to immune pathogenesis. To study the role of DC, biopsies from human skin were incubated with Bb and investigated in toto with electron microscopy. In addition, DC freshly isolated from dermis (DDC) and epidermis (Langerhans cells) were compared with blood-derived DC (BDC). In situ, Bb were found in the dermal layer of the skin only, occasionally cleared by DDC. Isolated DDC and BDC, but not Langerhans cells, readily engulfed Bb preferentially using coiling phagocytosis. Internalized Bb were located free in the cytosol and inside of phagolysosomes of DDC and BDC. Intravesicular Bb Ags were colocalized with MHC class II molecules. In addition, live Bb induced IL-12 production in BDC. Bb-pulsed BDC activated naive and primed autologous Bb-specific T cells, as measured by detection of granulocyte-macrophage CSF gene transcription and proliferative response, respectively. These data indicate that human DC phagocytose, process, and present Bb Ags. The way in which DC may influence the immune response in Lyme disease, however, remains to be evaluated.

Borrelia burgdorferi-induced ultrastructural alterations in human phagocytes: a clue to pathogenicity?

A chronic infection with the spirochaete Borrelia burgdorferi typically results in a multistage, multisystem illness. Thus, Lyme borreliosis may provide an interesting model to study the pathomechanisms of microbial persistence. In the present investigation, human peripheral blood monocytes, polymorphonuclear leukocytes, and synovial macrophages were incubated with B. burgdorferi and examined by light and electron microscopy. It was found that incubation with the spirochaetes induced distinct features in the phagocytes. Features which may be related to the pathogenesis of Lyme disease included the segmental uptake of spirochaetes with leaky lysosomes, the invagination of large membrane areas, the extra-lysosomal degradation of internalized B. burgdorferi cells and, finally, the formation of mononuclear syncytial cells and homotypic cell clusters. Features of unknown relevance were the occurrence of two types of cytoplasmic inclusion bodies and exocytic vesicles. These novel findings suggest that reactive alterations of the phagocytes may contribute to the pathogenesis of Lyme borreliosis, which could help to focus future histopathological studies. Moreover, these results may provide new insights into the pathogenesis of other infectious diseases characterized similarly by microbial persistence.