Enhanced expression of Toll-like receptors 2 and 4, but not 9, in spleen tissue from patients with visceral leishmaniasis.

Toll-like receptor (TLR) signalling is involved in first-line defence against Leishmania parasites by triggering NF-κB activation and downstream production of proinflammatory cytokines. Experimental models of visceral leishmaniasis (VL) support a protective role for TLRs 2, 4 and 9 in host immune responses to Leishmania infection. There are limited data available on expression of these TLRs in human VL, particularly in sites of infection, such as the spleen. This study aimed to determine whether the expression of mRNA encoding the expression of TLRs 2, 4 and 9 was altered in VL and compare expression patterns in splenic biopsies and peripheral blood mononuclear cells.

Tissue damage and immunity in cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is caused by parasitic infection of dermal macrophages resulting in intense immune-mediated tissue inflammation and skin ulceration. The severity of the disease is dependent on parasite species as well as the immune responses evoked by the host. Most cases of CL heal spontaneously. In rare cases, the ulcer/s become chronic, and some Leishmania species may induce mucosal leishmaniasis (MCL) leading to severe tissue damage. Due to difficulties in obtaining skin tissue, most human studies of CL have been limited to the analysis of peripheral blood. While systemic responses may be good correlates of immunity, tissue damage and local immune responses at the site of infection is seldom reflected in alterations in the peripheral blood. In this review, we discuss the different forms of CL focusing on the in situ responses in established disease and the mechanisms involved in pathology and healing of Leishmania infection. Great effort has been put into animal models dissecting the immune events behind the evolution of disease, tissue pathology and parasite control. These models of genetically engineered, immune deficient mice or mice given therapy prior to onset of overt disease poorly reflect the clinical situation, where patients seek treatment once infection is well established. Models of established disease are needed to address the clinical challenge of identifying new therapeutic targets in treatment CL. Through understanding immune deviations during CL potential benefits and risks of emerging biological drugs in leishmaniasis can be addressed.

Contribution of human neutrophils in the development of protective immune response during in vitro Leishmania major infection.

Stimulation of neutrophils may potentiate immunity to Leishmania major. CpG-containing oligodeoxynucleotide (ODN) has immune stimulatory effects and has been suggested as adjuvants and therapeutics to potentiate efficacy of vaccines and treatments against leishmaniasis. Here, we examined the stimulatory effect of synthetic ODN containing CpG motifs class A and B on cytokine production by neutrophils. Neutrophils from healthy donors responded to CpG-ODN type A, but not to class B, with secretion of IL-8 and following GM-CSF pretreatment with TNF-α production. To test whether neutrophil responses were altered in cutaneous leishmaniasis (CL) and to better understand the role of neutrophils in susceptibility and resistance to disease, we evaluated cytokine responses in GM-CSF preconditioned neutrophils from asymptomatic (Leishmanin skin test positive, LST+) and nonhealing CL individuals to CpG-ODN class A and assessed the expression levels of toll-like receptors (TLR2), 4 and 9. LST+ and healthy donor, but not nonhealing CL neutrophils, responded with TNF-α secretion. Neutrophils from nonhealing CL displayed increased mRNA expression levels of TLR2, 4 and 9 compared to neutrophils from LST+ or healthy donors. Therefore, failure to cure CL is associated with reduced ability of neutrophils to secrete TNF-α and correlates with high TLR 2, 4 and 9 expressions.

Human visceral leishmaniasis is not associated with expansion or accumulation of Foxp3+ CD4 cells in blood or spleen.

Natural regulatory T cells (CD4(+) CD25(+) Foxp3(+)), natural regulatory T cells (nTreg), play an important role in the regulation of inflammatory immune responses. However, the immunosuppressive properties of nTreg may unfavourably affect the host's ability to clear certain infections. In human visceral leishmaniasis (VL), reports on the frequency and function of nTreg are not conclusive. A limitation of our own previous studies that did not indicate a major role for Foxp3(+) nTreg in VL pathogenesis was that Foxp3 was measured by mRNA expression alone, as other tools were not available at the time. We have in this study assessed CD4(+)CD25(+)Foxp3(+) cells in splenic aspirates and peripheral blood mononuclear cells (PBMC) from an extensive series of patients with VL and endemic controls (EC) by flow cytometry (FACS). The results do not show increased frequencies of Foxp3(+) cells in patient with VL pre- and post-treatment, neither were they elevated when compared to PBMC of EC. We conclude that active VL is not associated with increased frequencies of peripheral Foxp3 Treg or accumulation at the site of infection.

Leishmania surface protein gp63 binds directly to human natural killer cells and inhibits proliferation.

Natural killer (NK) cells contribute to immunity as the first line of defence in numerous infections by early cytokine secretion and cytotoxicity. In Leishmania infection, NK cells contribute with interferon-gamma and may assist in directing the immune response towards T helper type 1, which is essential for successful control of the parasites. Thus, NK cells may play an important role in both resistance and control of the infection. However, during Leishmania infection NK cells show signs of suppression. To explore the reason for this suppression, we exposed naive and interleukin (IL)-2 activated NK cells directly to promastigotes of Leishmania major in vitro. As a rapid consequence of contact between naive NK cells and promastigotes, expression of NK cell receptors show significant changes. We identify one of the major surface molecules of promastigotes, glycoprotein (gp) 63, as an important agent for these suppressive effects by using promastigotes of a gp63ko strain of L. major. Furthermore, proliferation of IL-2-activated purified NK cells is suppressed after exposure to the wild-type but not to gp63ko promastigotes. However, gp63ko L. major induced no NK cell proliferation when NK cells were co-cultured with peripheral blood mononuclear cells populations such as CD14(+) monocytes or T cells.

Leishmanial amastigote antigen P-2 induces major histocompatibility complex class II-dependent natural killer-cell reactivity in cells from healthy donors.

Innate mechanisms involving natural killer cells have been implied to play an important role in immunity against Leishmania infection. Previous studies have evaluated responses to three purified amastigote antigens, P-2, P-4 and P-8, of Leishmania pifanoi. The P-4 and P-8 antigens have been demonstrated to induce protection in mouse models, as well as to induce cellular responses in American cutaneous leishmaniasis patients. Cells from Leishmania aethiopica-infected leishmaniasis patients preferentially responded to P-8 and, to a lesser extent, to the cysteine proteinase, P-2. In this study, it is shown that cells from healthy donors, including cells from truly naïve donors (cord blood), could be stimulated to proliferation and cytokine production by P-2. The main proliferating cell types in healthy adult donors were CD16/56(+) and the CD8(+) cells. Blocking of major histocompatibility complex (MHC) class II with alpha-MHC class II antibodies markedly inhibited proliferation and interferon-gamma (IFN-gamma) production, whereas interleukin-10 production was not affected. Experimental evidence indicates that CD4(+) cells were not necessary for the proliferative and IFN-gamma responses; however, an adherent cell population was required. Furthermore, CD16/56(+) cells expressing MHC class II were expanded following P-2 stimulation. The responses to P-2 show a striking similarity to responses induced by the vaccine candidate Leishmania homologue of receptors for activated C-kinase (LACK) in healthy donors. The responses described here may not be desirable when aiming at inducing protective immune responses with a vaccine, and the implications of these results for the development of vaccines against leishmaniasis are discussed.

Live Leishmania promastigotes can directly activate primary human natural killer cells to produce interferon-gamma.

Natural killer (NK) cells have been implicated in the natural protection and healing of leishmaniasis by their ability to secrete the macrophage activating cytokine interferon (IFN)gamma. Previous studies have demonstrated that early production of interleukin (IL)-12 triggers IFN gamma secretion by NK cells. Here we report that live Leishmania promastigotes (the form that is injected by the vector) can directly induce human peripheral blood NK cells from healthy donors to IFN gamma secretion in the absence of IL-12 and professional antigen presenting cells. Killing of promastigotes abolishes this property. This novel mechanism of activation of the innate immune response may be relevant for establishment of infection and thus also the design of vaccines against leishmaniasis.

Differential induction of cellular responses by live and dead Leishmania promastigotes in healthy donors.

The most effective protection against human leishmaniasis has been achieved following vaccination with live promastigotes. Killed promastigotes + BCG can protect, albeit to a lower degree. To explore what mechanisms may be involved in these differences, the ability of live and dead promastigotes to induce immune responses were evaluated in vitro. The data showed that live and dead promastigotes differ in their ability to induce proliferation and cytokine production. Cytokine gene expression of Th1 related cytokines (IL-12, IFNgamma and TNFalpha) in adult PBMC was more evident to live than to heat killed promastigotes. This was coupled with significantly higher number of IFNgamma secreting cells induced by live than killed promastigotes. However, alpha-IL-12 antibodies did not block the IFNgamma response induced by live promastigotes. Proliferative responses were variable. In contrast to adult PBMC no IFNgamma secreting MNC could be detected in cord blood. However, in these cells the live promastigotes consistently induced higher proliferative response compared to dead. Implications of these findings are discussed.

A Leishmania homologue of receptors for activated C-kinase (LACK) induces both interferon-gamma and interleukin-10 in natural killer cells of healthy blood donors.

Natural killer (NK) cells from individuals unexposed to Leishmania organisms proliferate with high interferon (IFN)-gamma secretion in response to crude Leishmania antigen preparations. In an attempt to identify the molecules that induce blood cells to proliferate and to secrete cytokines, we tested the effect of a 36-kDa Leishmania homologue of receptors for activated C-kinase (LACK) on peripheral blood mononuclear cells from unexposed individuals. Mainly CD8(+) and NK cells proliferated in response to LACK. At both the mRNA and soluble protein level, the main sources for LACK-induced IFN-gamma and interleukin (IL)-10 were T and NK cells. Furthermore, in the presence of anti-major histocompatibility complex (MHC) class II antibody, there was inhibition of LACK responses in both CD4(+) and CD16/56(+) cells, with a marked decrease in IFN-gamma but with an increase in IL-10 production. We conclude that the response to LACK is part of the response to Leishmania organisms in unexposed donors described elsewhere. That this NK-dominated response is MHC class II sensitive, whether through a direct or indirect effect, is discussed.

Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis.

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.

Natural killer cells in cross-regulation of IL-12 by IL-10 in Leishmania antigen-stimulated blood donor cells.

We have previously shown that natural killer (NK) cells play a role in protection against leishmaniasis. Furthermore, we have shown that NK cells in mononuclear cells derived from unexposed donors are induced to proliferate in vitro in response to leishmanial antigens. Since interleukin (IL)-12, a strong inducer of NK cells, acts on the early events in NK cells and T-cells, and is considered as an adjuvant for use in a potential antileishmaniasis antigen, we wished to investigate how this cytokine influences the in vitro Leishmania induced proliferative and cytokine response in healthy donors. We demonstrate that in an innate response to Leishmania antigen involving NK cells, a critical level of IL-12 is required to induce interferon (IFN)-gamma secretion below which, IL-10 is released in amounts which apparently inhibit IFN-gamma secretion and cellular proliferation. However, at higher IL-12 levels, there is simultaneous secretion of IFN-gamma and IL-10 as well as proliferation of cells. In a similar vein, exogenous IL-10 in turn inhibited IFN-gamma secretion as well as proliferation when used at low/medium concentrations, but at high concentrations this effect was abolished and replaced by the simultaneous detection of IFN-gamma, IL-10 and proliferation. The contribution of NK cells in cross regulation of these two very important immuneregulatory cytokines and the effect of exogenous IL-12 in a Leishmania driven response are discussed.

Trapeziectomy and ligament reconstruction for osteoarthrosis of the base of the thumb. A prospective study of 100 operations.

100 thumbs with primary osteoarthrosis of the joints of the trapezium were treated by trapeziectomy and a FCR sling arthroplasty to reconstruct a first intermetacarpal ligament by the method described by Burton and Pellegrini (1986). Pain at rest remained in five. Some pain at or after exertion persisted in 46, and 49 became completely pain-free. 88 were satisfied with the procedure and there was a significant increase in pinch strength and in the ability to perform activities of daily life. It has become our preferred procedure for treating osteoarthrosis of the basal joint of the thumb.

Weilby tendon interposition arthroplasty for osteoarthritis of the trapezial joints.

Primary osteoarthritis of the trapezial joints has been treated by an interposition tendoplasty according to Weilby in eighty-nine cases. After excision of the trapezium, a strip from the flexor carpi radialis was wound around the main portion of the flexor carpi radialis tendon and the abductor pollicis longus. The abductor tendon was then duplicated over the tendoplasty and reinserted to the first metacarpal base. In 40% of cases, osteoarthritis was present in more than one trapezial joint. 57% had an adduction contracture of the first metacarpal, half of which were relieved postoperatively. 73% of patients were satisfied at follow-up. Complications included four cases with loss of active metacarpal abduction which was regained after reinsertion of the abductor pollicis longus. It is concluded that the Weilby tendoplasty is a useful alternative to Silastic implants, especially in cases of adduction contracture. The risks of implant dislocation and silicone synovitis are eliminated.

Swanson implant arthroplasty of the wrist in rheumatoid arthritis.

Sixty rheumatoid wrists operated with Swanson implant arthroplasty were evaluated after a mean observation time of thirty-three months. Grip function in daily living improved in 60%, pain decreased in 88%, range of motion increased in 83% and grip strength increased in 69% of operated wrists. Significantly impaired function was found in wrists with implant fracture (12%) and in cases with pronounced bone resorption around the implant (23%). Ulnar deviation and carpal collapse were commonly found but did not impair the function significantly. In seven patients the contralateral wrist had been fused and was compared to the arthroplasty. The merits and indications of arthroplasty are discussed.

Time factor, infection frequency and quantitative microbiology in hand injuries: a prospective study.

In a prospective study for 108 surgically treated hand wounds the infection rate was significantly lower in tidy than in untidy injuries (6 and 32%). Infection was significantly more frequent when slough developed than in wounds without slough (54 and 11%). Only seven patients received antibiotic prophylaxis. Quantitative analysis of bacteria in the wound margins was performed in 89 cases. No significant difference in the frequency of infection was found when the cultures yielded greater than 10(5) or less than 10(5) bacteria/gram tissue. Prolongation of the interval between injury and operation (up to 18 hours) was not associated with increased rate of infection. Cases with longer intervals than 18 hours were too few for statistical analysis. The duration of the operation or of tourniquet application did not influence the infection rate. The importance of adequate wound care, gentle technique and staged treatment are discussed, and also the question of prophylactic antibiotic medication.

Induced fat embolism in rabbits: effects of defibrinogenation and thrombocytopenia.

Fat embolism was induced in rabbits by giving an intravenous injection of radioactively labelled homologous retroperitoneal fat. One group was defibrinogenated by Arvin. A second group was made thrombocytopenic by treatment with Busulphan. After the animals had been made thrombocytopenic and had been defibrinogenated, respectively, fat embolism was induced. Macroscopic and histologic examinations were carried out, as well as recordings of wet weight and radioactivity content of the lung. There was a highly significant increase in the pulmonary wet weight of thrombocytopenic animals compared with controls. Macroscopic as well as histologic examination revealed a massive interstitial and alveolar oedema. Half of the specimens showed moderate to massive bleedings. Defibrinogenated animals did not differ from controls. Under the given circumstances, the results suggest that platelets are protective to the endothelial lining of the pulmonary capillaries during embolism. The presence or absence of fibrinogen initially does not seem to be of major importance for the pulmonary damage induced by fat injection.