Antileishmanial defense in macrophages triggered by tumor necrosis factor expressed on CD4+ T lymphocyte plasma membrane.

In our studies of host defense to the intracellular protozoan Leishmania major, we uncovered a novel mechanism of antileishmanial defense that involves direct cell contact between effector CD4+ lymphocytes and Leishmania-infected macrophages. The mechanism is distinctive because it does not involve lymphokine secretion and induces no cytotoxic effects in the host cells; its expression is antigen-specific and genetically restricted. We now demonstrate that these effector CD4+ cells display tumor necrosis factor (TNF) on their surface and provide evidence that the membrane-associated TNF is involved in the activation of the antileishmanial defense. Using a Leishmania-specific cloned T-T cell hybridoma line (1B6; CD4+, T helper type 1) that activates antileishmanial defense in macrophages through cell contact and does not secrete TNF, we noted that only cells bearing surface TNF (TNF+), but not ones lacking surface TNF (TNF-), exerted these effects. Moreover, the antileishmanial effects excreted by TNF+ 1B6 cells as well as by lymph node CD4+ TNF+ lymphocytes could be blocked with anti-TNF antibody. We propose that membrane-associated TNF on CD4+ T cells may provide a mechanism of targeting activation signals to macrophages in an antigen-specific and genetically restricted manner.

Hepatic fibrosis in schistosomiasis: egg granulomas secrete fibroblast stimulating factor in vitro.

Cytosol extracts and culture supernatants of isolated egg granulomas obtained from livers of mice with Schistosoma mansoni infection stimulated fibroblasts to incorporate tritiated thymidine and to proliferate in vitro. This finding suggests that hepatic granulomas may play a role in regulating hepatic fibrosis in Schistosoma mansoni infections.

In vitro parasite-monocyte interactions in human leishmaniasis. Evidence for an active role of the parasite in attachment.

Leishmania are obligate intracellular parasites of mononuclear phagocytes in the mammalian host. To more clearly define features of the early events in host-parasite interaction, human monocytes were so-cultured with Leishmania tropica amastigotes in vitro. Infection of monocytes was time dependent and inhibited at 4 degrees C and in the presence of cytochalasin B. Pretreatment of amastigotes with cytochalasins prevented their attachment to normal monocytes. Untreated amastigotes attached normally but could not enter cytochalasin-pretreated monocytes. This suggests that amastigotes actively participate in attachment but require host cell participation for interiorization.

Intravascular clearance of parasitized erythrocytes in rodent malaria.

Little is known about host defense mechanisms responsible for protective immunity in malaria. The intravascular location of the infection suggested that removal of parasitized erythrocytes by reticuloendothelial organs might be important. To study this possibility, we examined the clearance of (51)Crlabeled Plasmodium berghei-infected erythrocytes in rats. Infected erythrocytes were removed more rapidly from circulation than homologous uninfected erythrocytes. The rate of clearance of infected cells during the 1st hour after inoculation was approximately three times greater in rats rendered immune by prior infection than in control rats. This accelerated clearance resulted from greater splenic uptake in immune rats and appeared to correlate with spleen size. Since the clearance pattern of infected erythrocytes more closely resembled the clearance of Heinz body-containing uninfected erythrocytes than of antibody-coated (immunoglobulin G) uninfected erythrocytes, rheologic alterations of parasitized erythrocytes might be a more important determinant of clearance than an antibody-dependent process. During the phase of malaria infection in which increasing parasitemia is observed, organ uptake of infected erythrocytes did not increase despite splenic and hepatic enlargement. However during the spontaneous onset of resolution of malaria infection characterized by decreasing parasitemia, a marked enhancement of splenic clearance was noted. These observations suggest that sudden alteration in splenic clearance of parasitized erythrocytes might be important in the resolution of acute malaria.

Relationship of alterations in splenic clearance function and microcirculation to host defense in acute rodent malaria.

During the course of Plasmodium berghei malaria in the rat, splenic clearance of damaged uninfected erythrocytes (heated or Heinz body-containing) underwent changes strikingly similar to those of infected erythrocytes. Splenic trapping of abnormal erythrocytes was impaired during the period of rising parasitemia but became supernormal just before the onset of resolution of the acute infection. These changes could be related to the development of splenomegaly and alterations in splenic cordal microcirculation during infection. The relative distribution of flow through the cords was decreased during rising parasitemia and was restored before the onset of resolution. Together, our observations support the hypothesis that altered rheologic properties of infected erythrocytes are a major determinant of their removal by the spleen. These data suggest that the alterations in splenic microcirculation that occur during malaria may have important implications for host defense.

Sildenafil improves clinical signs and radiographic features in dogs with congenital idiopathic megaoesophagus: a randomised controlled trial.

We evaluated the efficacy of oral sildenafil citrate in dogs with congenital idiopathic megaoesophagus (CIM). Twenty-one puppies were randomly assigned to two groups (treatment and control). The dogs were given sildenafil oral suspension 1 mg/kg every 12 hours for 14 days or placebo in a masked fashion. Clinical signs (frequency of regurgitation and weight gain) and oesophagrams (relative oesophageal diameter, ROD) were evaluated in order to assess the efficacy of drug treatment, by examiners who were unaware of the study protocol. In addition, a set of in vitro experiments on isolated samples of canine lower oesophageal sphincter (LOS) was performed, and the effects of increasing concentrations of sildenafil on basal tone and electrically-stimulated motility were assessed. Sildenafil administration significantly reduced the number of regurgitation episodes (0.88±1.40 v 2.65±1.56, P<0.0001) and significantly increased weight gain in the treated dogs compared to controls (79.76±28.30 per cent v 53.40±19.30 per cent, P=0.034). ROD values, at the end of the treatment period, were significantly decreased in the sildenafil group, compared to pre-treatment values (0.97±0.19 v 0.24±0.14, P<0.0001), in contrast to control subjects (0.98±0.17 v 1.10±0.25, P=0.480). In accordance with the in vivo findings, sildenafil dose-dependently reduced basal tone and increased electrically-induced relaxation of dog LOS samples. These results suggest that sildenafil citrate helps ameliorate clinical and radiographic signs in dogs with CIM by reducing LOS tone, and could represent a novel therapeutic tool for the treatment of this disease.

An Acute Ocfentanil Fatality: A Case Report with Postmortem Concentrations.

A 24-year-old man known to consume illegal drugs was found dead in his apartment. A reclosable plastic zipper bag containing several hundred milligrams of a brown powder was found close to the dead body and the first assumption of the investigators was death due to heroin intoxication. Therefore, a legal autopsy was ordered. The following toxicological analysis revealed ocfentanil in urine and in the brown powder. Four different approaches for the determination of the ocfentanil concentrations in peripheral whole blood are described. Enrichment of ocfentanil from the powder was realized. With this reference, it was possible to determine the ocfentanil concentration in the seized powder to be 0.91%. Concentrations of ocfentanil were also determined in the sampled body fluids using the standard addition procedure. In peripheral blood 9.1 µg/L, in heart blood 27.9 µg/L and in urine 480 µg/L were measured. In addition, the antidepressant citalopram, the neuroleptic quetiapine and cannabinoids were found in urine and subsequently quantified in peripheral blood.

Soluble TNF and membrane TNF expressed on CD4+ T lymphocytes differ in their ability to activate macrophage antileishmanial defense.

In our studies of host defense against the intracellular parasite Leishmania major, we obtained evidence for a novel mechanism of macrophage activation for antimicrobial defense that involves direct cell contact between CD4+ T lymphocytes and Leishmania-infected macrophages. The mechanism is distinctive as it does not involve secretion of lymphokines but is apparently mediated by the membrane-anchored form of tumor necrosis factor (mTNF; approximately 50-60 kd) present on the surface of the effector T lymphocytes. Furthermore, it is not cytotoxic to the host cell and its expression is antigen specific and genetically restricted. We prepared a Leishmania-specific cloned T-T cell hybridoma line 1B6 (CD4+, TH1) that expresses membrane-bound TNF but does not secrete TNF or other macrophage activators. We now report that 1B6 cells can activate antileishmanial defense in inflammatory macrophages, whereas soluble recombinant murine TNF (sTNF) alone is unable to do so. On the other hand, both 1B6 cells and sTNF can act synergistically with recombinant murine interferon-gamma (IFN-gamma, a known soluble macrophage-activating factor) in activating antimicrobial defense and NO2- release. The effects of 1B6 alone and the synergistic effects of 1B6 and IFN-gamma or sTNF and IFN-gamma are arginine dependent. These results suggest that mTNF may be more efficient than sTNF in macrophage activation and that contact with effector CD4+ lymphocytes that express mTNF may be an important mechanism of host defense.

[Examination of victims of sexual violence--the problem of false reports]. / Untersuchungen von Opfern sexueller Gewalt- das Problem der Falschanzeige.

Some people claim to be victims of sexual violence and abuse the judicial system by filing a false police report. Generally, motivation for such behaviour is assumed to be that the self-proclaimed victims try to avoid taking responsibility for a sexual act in which they have been an active, willing participant. Concise motivations include attempts to mask a sexual affair from a partner, or to mask a first sexual intercourse by pretending to be the victim of an act of violence. By the nature of many judicial systems that are abused for such purposes, clinical forensic specialists are confronted with such cases far more often than clinical doctors not serving judicial authorities. In adults, history, injuries and other findings often show patterns that can be recognized as evidence of deception or as signs for self-inflicted injury for the trained specialist. This is far less often the case in infants and children.

DNA synthesis in promastigotes of Leishmania major and L. donovani.

The requirement of protozoan parasites for pre-formed purines affords the opportunity for quantitation of nucleic acid synthesis from incorporation of radioactively labeled purines into DNA and RNA. We have developed rapid and simple assays to quantitate DNA and RNA synthesis in promastigotes of Leishmania major and L. donovani from the incorporation of [3H]hypoxanthine. DNA but not RNA synthesis in L. major or L. donovani promastigotes was inhibited by aphidicolin (50% inhibition by 0.2-0.3 microM) and by hydroxyurea (50% inhibition by 0.3-0.5 mM). The inhibition of DNA synthesis by aphidicolin or hydroxyurea was reversible when the inhibitor was removed 2, 4 or 24 h after its addition. Several well-characterized agents that inhibit DNA synthesis in mammalian cells, 1-beta-D-arabinofuranosylcytosine (araC), 9-beta-D-arabinofuranosyladenine (araA), phosphonoacetic acid, novobiocin and N2-(p-n-butylphenyl)guanine (BuPG), failed to inhibit DNA synthesis in promastigotes of L. major even when used at very high concentrations, demonstrating differences between DNA replication components of parasite and host.

A quantitative assay to detect circulating fibrosin and its application in experimental schistosomiasis.

The molecular pathogenesis of schistosomal liver fibrosis has been studied in a murine model of Schistosoma mansoni. A novel fibroblast growth factor, fibrosin, was identified as a product of a subpopulation of CD4+ lymphocytes resident in the hepatic egg granulomas. We describe a sensitive, quantitative, antigen-capture enzyme-linked immunosorbent assay that can detect fibrosin in mouse serum and plasma at concentrations > 0.05 pg/ml. Using this assay, we detected in infected mice circulating fibrosin levels that were several-fold increased above those detected in uninfected controls. Circulating fibrosin levels increased after week 4 of infection, reached a peak at week 8, and normalized by week 12. We propose that circulating fibrosin might be a useful marker of hepatic fibrogenesis in schistosomiasis.

Resolution of acute malaria (Plasmodium berghei in the rat): reversibility and spleen dependence.

Six-week-old rats infected with Plasmodium berghei developed a peak parasitemia of 55.2 +/- 3.1% by day 15 of infection, followed by spontaneous resolution of the infection during a process referred to as crisis. Crisis was accompanied by the appearance in circulation of infected erythrocytes in which the parasites appeared abnormal ("crisis forms"). Rats splenectomized at different times during the crisis period experienced a sudden increase in parasitemia, with a marked decrease in the number of circulating crisis forms. Splenectomy in parasitemia, with a marked decrease in the number of circulating crisis forms. Splenectomy resulted in a 100% mortality, whereas all control and sham-operated rats survived their infection. Although P. berghei is restricted to developing within young erythrocytes, our observations could not be explained by the effects of splenectomy on the number of circulating reticulocytes. Indeed, the reticulocytosis which accompanies crisis was unaffected by splenectomy. Our observations therefore suggest that crisis is a reversible process and, specifically, that the spleen is necessary for its maintenance.

Cutaneous leishmaniasis--a case with persistent organisms after treatment in presence of normal immune response.

A Peace Corps volunteer in Senegal, West Africa contracted cutaneous leishmaniasis which had several noteworthy features. One of the three presenting cutaneous ulcers was associated with subcutaneous nodules and viable organisms were recovered from healing lesions after multiple courses of treatment, including amphotericin B. Yet, the patient was found to exhibit both humoral and cell mediated features of normal immunologic responsiveness. Ultimately, clinical and parasitological cure occurred. The patient's organism was found to produce lesions in the foot pads of mice.

Malaria in asplenic mice: effects of splenectomy, congenital asplenia, and splenic reconstitution on the course of infection.

In investigations on the role of the spleen in host defense against malaria, we studied the course of murine malaria in three groups of mice with altered splenic function: congenitally asplenic mice, adult-splenectomized mice, and adult-splenectomized mice which were reconstituted with spleen-cell suspensions. Intact mice infected with either Plasmodium yoelii or P. chabaudi adami experienced infections which resolved spontaneously, with low mortality. Congenitally asplenic and splenectomized-reconstituted mice were unable to clear their primary infections, and experienced high mortality; infections in the latter two groups of mice differed little from those in splenectomized, nonreconstituted controls. However, when asplenic and splenectomized mice were treated with cloroquine during their primary infections and then rechallenged with the homologous Plasmodium species, they experienced mild infections similar to those of intact controls. These observations support the concept that the host defense in primary malaria infections requires an architecturally intact spleen, and therefore is not solely dependent upon the presence of a subpopulation of immune spleen cells.

Cellular hypersensitivity to toxoplasmal and retinal antigens in patients with toxoplasmal retinochoroiditis.

To investigate the possible role of hypersensitivity to toxoplasmal and retinal antigens in patients with toxoplasmal retinochoroiditis, we examined their in vitro lymphoproliferative responses to antigens prepared from Toxoplasma gondii and human retina. The magnitude of patients' responses, determined by incorporation of [3H]-thymidine, was compared to those of Toxoplasma seropositive and seronegative controls. Patients were indistinguishable from seropositive controls in terms of antitoxoplasmal antibody titer (dye test, indirect hemagglutination and enzyme-linked immunosorbent assay) and in vitro lymphoproliferative responses to toxoplasmal antigens. Furthermore, there was no relationship between antibody titer and the magnitude of proliferative responses in seropositive individuals. Four of four patients with active eye disease and six of 13 with inactive disease, but none of the seropositive or seronegative controls, had significant lymphoproliferative responses to human retinal antigens. These observations raise the possibility of an autoimmune component in the pathogenesis of relapses in toxoplasmal retinochoroiditis.

Exogenous interferon administration in experimental leishmaniasis: in vivo and in vitro studies.

The potential feasibility of using exogenously administered human interferon for the treatment of selected cases of leishmaniasis prompted us to study the effects of murine interferon on the course of Leishmania tropica infection in C57Bl/6 mice. L cell-derived mouse interferon was administered daily by intraperitoneal (1,000 and 10,000 U) or intralesional (100 U) injection in mice inoculated into footpads with L. tropica amastigotes. Footpad swelling and tissue parasite density were assessed over the course of infection. Interferon treatment did not significantly affect these clinical and parasitological parameters. Furthermore, addition of interferon (100--100,000 U) to cultures of amastigote-infected mouse peritoneal macrophages or to axenic cultures of promastigotes did not affect replication. We conclude that interferon lacks intrinsic antileishmanial activity and does not significantly enhance host defense against Leishmania.

Circulating factor from a kala-azar patient suppresses in vitro antileishmanial T cell proliferation.

In vitro lymphocyte blastogenesis stimulated by phytohaemagglutinin (PHA), streptococcal antigens (SKSD) and leishmanial antigens were used to assess T cell responsiveness in one patient with kala-azar before and after therapy. During the illness, responses to PHA and SKSD but not to leishmanial antigens could be detected. After treatment lymphocytes responded to all three stimuli when cells were cultured in convalescent plasma, but failed to respond to leishmanial antigens when cultured in plasma obtained before treatment. These observations suggest the presence of a circulating inhibitor of anti-leishmanial T cell responsiveness in kala-azar, and warrant further investigation.

Human cutaneous leishmaniasis: in-vitro parasite--mononuclear cell interactions in immune and naive individuals.

The aim of this study was to compare the ability of Leishmania parasites to survive in mononuclear cells from immune individuals with their ability to survive in cells from naive individuals. For this purpose we established an in vitro system based on the co-culture in suspension of human peripheral blood leukocytes derived from immune and naive subjects and L. major promastigotes. The proportion of monocytes containing intracellular parasites and the number of amastigotes per 100 infected monocytes (parasite burden) were determined 24 and 72 h after in-vitro infection. The proportion of infected cells from naive individuals did not change, and the number of amastigotes either did not change or increased by 1.2 to 1.7-fold between 24 and 72 h incubation. In contrast, in the immune subjects, the proportion of infected monocytes 24 h after infection was lower than in the naive individuals, and a 30-90% decrease in both the proportion of infected monocytes and the parasite burden was observed after 72 h incubation. Based on these results, three characteristics of leishmanicidal activity of mononuclear cells from immune individuals were determined: (a) the proportion of infected monocytes 24 h after infection was lower than 22%; (b) there was a decrease of more than 30% in the proportion of infected monocytes between 24 and 72 h after infection; and (c) there was a significant decrease in the number of amastigotes between 24 and 72 h after infection. The results of this study demonstrate an enhanced leishmanicidal activity of mononuclear cells from immune individuals.

Fatal cardiac tamponade associated with posterior spinal instrumentation. A case report.

STUDY DESIGN: Case report of a fatal complication of pedicle screw instrumentation and review of the literature. OBJECTIVE: To describe the clinical and postmortem findings in a 35-year-old man who sustained a T11 burst fracture that was managed by transpedicular posterior instrumentation and who died 12 days after surgery of cardiac tamponade caused by a prick injury of the right coronary artery. SUMMARY OF BACKGROUND DATA: Posterior pedicle screw instrumentation is considered a safe and effective method for stabilizing a spinal motion segment. Nevertheless, there are several rare but significant complications that may occur. This is the first report of a heart tamponade after transpedicular screw insertion. METHODS: A 35-year-old man was treated for a T11 burst fracture with posterior transpedicular stabilization. The surgery was uncomplicated. RESULTS: Twelve days after the intervention, the patient died of cardiogenic shock. Postmortem examination showed a heart tamponade of 350 mL blood originating in a prick injury of the right coronary artery. Histologic findings showed evidence that the injury was caused during surgery by a Kirschner wire. CONCLUSION: There are numerous possible intraoperative complications in posterior pedicle screw fixation, such as nerve root and spinal cord injuries. This case of a fatal heart tamponade after transpedicular screw insertion is rare. It shows that the surgeon must be aware of potential risks associated with such a procedure and have a comprehensive three-dimensional understanding of the anatomic structures involved.

Splenic functions in malaria.

The spleen traps parasitized erythrocytes within its unique architecture, and this trapping function is modified during plasmodium infestation. Splenectomy dramatically exacerbates the course of experimental malarias probably through elimination of activated cordal macrophages responsible for killing intraerythrocytic plasmodia.