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Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.

Pneumococcal meningitis often results in death or neurological sequelae, but the underlying pathogenetic mechanisms remain poorly understood. In C57BL/6J mice subjected to intracerebroventricular (icv) challenge with Streptococcus pneumoniae, the chemokine CCL2 and cytokines interferon-γ, interleukin (IL)-1β, IL-6 and tumour necrosis factor were prominently expressed in the brain during the acute phase of the disease. The upregulation of these immune mediators was markedly diminished in IL-18-deficient mice. Uninfected IL-18(-/-) mice exhibited decreases in anxiety phenotype and licking behaviour, and an increase in behavioural habituation, in an automated monitoring system (the IntelliCage). Without antibiotic intervention, a majority of IL-18(+/+) mice developed irreversible disease after icv S. pneumoniae but this was significantly improved by deleting IL-18 gene function. IL-18(+/+) mice cured of pneumococcal meningitis with four doses of ceftriaxone, initiated at 20 h post-inoculation, showed enduring sequelae. These included abnormal behavioural phenotypes featuring diurnal hypoactivity and nocturnal hyperactivity, light phobia and disrupted cognitive function. While the hyperactive phenotype was absent in the corresponding IL-18(-/-) survivors, cognitive impairments and behavioural deficits were still present. Overall, the results suggest that the high levels of cytokines and/or chemokines released after pneumococcal challenge provoked a series of pathological events, ultimately causing acute death. Furthermore, since only a subset of behavioural phenotypes were ameliorated in the pneumococcus-infected IL-18(-/-) mice, the pathological pathways causing mortality may be, at least in part, distinct from those leading to long-term neurological sequelae.

Sequestration of infected red blood cells (iRBC) within the cerebral and pulmonary microvasculature is a hallmark of human cerebral malaria (hCM). The interaction between iRBC and the endothelium in hCM has been studied extensively and is linked to the severity of malaria. Experimental CM (eCM) caused by Plasmodium berghei ANKA reproduces most features of hCM, although the sequestration of RBC infected by P. berghei ANKA (PbA-iRBC) has not been completely delineated. The role of PbA-iRBC sequestration in the severity of eCM is not well characterized. Using static and flow cytoadherence assays, we provide the first direct in vitro evidence for the binding of PbA-iRBC to murine brain and lung microvascular endothelial cells (MVEC). We found that basal PbA-iRBC cytoadherence to MVECs was significantly higher than that of normal red blood cells (NRBC) and of RBC infected with P. berghei K173 (PbK173-iRBC), a strain that causes noncerebral malaria (NCM). MVEC prestimulation with tumor necrosis factor (TNF) failed to promote any further significant increase in mixed-stage iRBC adherence. Interestingly, enrichment of the blood for mature parasites significantly increased PbA-iRBC binding to the MVECs prestimulated with TNF, while blockade of VCAM-1 reduced this adhesion. Our study provides evidence for the firm, flow-resistant binding to endothelial cells of iRBC from strain ANKA-infected mice, which develop CM, and for less binding of iRBC from strain K173-infected mice, which develop NCM. An understanding of P. berghei cytoadherence may help elucidate the importance of sequestration in the development of CM and aid the development of antibinding therapies to help reduce the burden of this syndrome.

Type 2 immunity is critical for defense against cutaneous infections but also underlies the development of allergic skin diseases. We report the identification in normal mouse dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depended on interleukin 7 (IL-7) and constitutively produced IL-13. Intravital multiphoton microscopy showed that dermal ILC2 cells specifically interacted with mast cells, whose function was suppressed by IL-13. Treatment of mice deficient in recombination-activating gene 1 (Rag1⁻/⁻) with IL-2 resulted in the population expansion of activated, IL-5-producing dermal ILC2 cells, which led to spontaneous dermatitis characterized by eosinophil infiltrates and activated mast cells. Our data show that ILC2 cells have both pro- and anti-inflammatory properties and identify a previously unknown interactive pathway between two innate populations of cells of the immune system linked to type 2 immunity and allergic diseases. Refereed/Peer-reviewed

The effectiveness of praziquantel (Droncit) against yellow grubs Clinostomum complanatum and unidentified, encysted, larval trematodes was tested in infected sunshine bass Morone chrysops female × M. saxatilis male. Praziquantel treatments significantly reduced the total number of live grubs in the fish in most treatment regimes tested. Treatment at 0.25 mg/L for 24 h was as effective as one at 8 mg/L for 8 h. Mature dead and dying metacercaria may have released toxins that killed host fish in some treatments.

We investigated the relationship between gut health, visceral fat dysfunction and metabolic disorders in diet-induced obesity. C57BL/6J mice were fed control or high saturated fat diet (HFD). Circulating glucose, insulin and inflammatory markers were measured. Proximal colon barrier function was assessed by measuring transepithelial resistance and mRNA expression of tight-junction proteins. Gut microbiota profile was determined by 16S rDNA pyrosequencing. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNA levels were measured in proximal colon, adipose tissue and liver using RT-qPCR. Adipose macrophage infiltration (F4/80+) was assessed using immunohistochemical staining. HFD mice had a higher insulin/glucose ratio (P = 0.020) and serum levels of serum amyloid A3 (131%; P = 0.008) but reduced circulating adiponectin (64%; P = 0.011). In proximal colon of HFD mice compared to mice fed the control diet, transepithelial resistance and mRNA expression of zona occludens 1 were reduced by 38% (P

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with E columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean +/- SE) was noted between the channel catfish in treatment 1 (95.2 +/- 1.2%) and those in treatment 2 (95.6 +/- 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 + 4.5%) and 4 (41.8 +/- 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 x 10(5)) than did treatment 3 fish (1.2 x 10(7)), and treatment 2 fish (8.5 x 10(3)) had significantly less bacteria than did treatment 1 fish (6.9 x 10(4)), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 x 10(7)) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F columnare.

Background Group 2 innate lymphoid cells (ILC2) have been implicated in the pathogenesis of allergic lung diseases. However, the upstream signals that regulate ILC2 function during pulmonary inflammation remain poorly understood. ILC2s have been shown to respond to exogenous IL-2, but the importance of endogenous IL-2 in ILC2 function in vivo remains unclear. Objective We sought to understand the role of IL-2 in the regulation of ILC2 function in the lung. Methods We used histology, flow cytometry, immunohistochemistry, ELISA, and quantitative PCR with knockout and reporter mice to dissect pulmonary ILC2 function in vivo . We examined the role of ILC2s in eosinophilic crystalline pneumonia, an idiopathic type 2 inflammatory lung condition of mice, and the effect of IL-2 deficiency on this disease. We determined the effect of IL-2 administration on pulmonary ILC2 numbers and function in mice in the steady state and after challenge with IL-33. Results We discovered an unexpected role for innate cell–derived IL-2 as a major cofactor of ILC2 function during pulmonary inflammation. Specifically, we found that IL-2 was essential for the development of eosinophilic crystalline pneumonia, a type 2 disease characterized by increased numbers of activated ILC2s. We show that IL-2 signaling serves 2 distinct functions in lung ILC2s, namely promoting cell survival/proliferation and serving as a cofactor for the production of type 2 cytokines. We further demonstrate that group 3 innate lymphoid cells are an innate immune source of IL-2 in the lung. Conclusion Innate cell–derived IL-2 is a critical cofactor in regulating ILC2 function in pulmonary type 2 pathology.

N/A NHMRC

Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca2+ chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na+/H+ exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions. © 2011 The Authors. Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

The autofluorescence of myeloid cell populations is heterogeneous and can be used as a tool for identification and phenotyping of myeloid subsets. Cellular AF is usually considered a hindrance to flow cytometric analysis. Here, we incorporate AF into analysis of complex mixtures of leukocytes. Using a mouse model, we examined cellular AF at multiple excitation and emission wavelengths, and populations with discrete patterns were gated and examined for surface marker expression. In the spleen, all major myeloid populations were identified. In particular, the approach allowed simultaneous characterization of RPM and resident monocytes. When monocytes and RPM were compared, RPM exhibited a phenotype that was consistent with involvement in physiological processes, including expression of genes involved in lipid and iron metabolism. The presence of large amounts of stored ferric iron within RPM enabled purification of these cells using a magnetic-based approach. When adapted for use on leukocytes isolated from a range of other organs, incorporation of AF into analysis allowed identification and isolation of biologically important myeloid populations, including subsets that were not readily identifiable by conventional cytometric analysis.

Cerebral malaria (CM) is a fatal complication of Plasmodium falciparum infection. Using a well defined murine model, we observed the effect on disease outcome of temporarily reducing parasite burden by anti-malarial drug treatment. The anti-malarial treatment regime chosen decreased parasitaemia but did not cure the mice, allowing recrudescence of parasites. These mice were protected against CM, despite their parasitaemia having increased, following treatment cessation, to levels surpassing that associated with CM in mice not treated with the drug. The protection was associated with reduced levels of cytokines, chemokines, CD8+ T cells and parasites in the brain. The results suggest that the development of the immunopathological response that causes CM depends on a continuous stimulus provided by parasitised red blood cells, either circulating or sequestered in small vessels. © 2010 Australian Society for Parasitology Inc.

The efficacy of potassium permanganate (KMnO4) against the early stages of an experimental acute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus, was evaluated. Fish were experimentally challenged by waterborne exposure for 2 h to F. columnare after cutaneous abrasion, and treated with KMnO4 at 2.0 mg L−1 above the KMnO4 demand at 0, 1, 2 or 4 h postchallenge for 24 h. Challenged non-treated fish acted as a positive control and non-challenged non-treated fish acted as a negative control. Fish challenged and treated with KMnO4 at 0, 1, 2 or 4 h postchallenge had mortalities of 26%, 63%, 64% and 83% respectively. The mortality of challenged fish treated with KMnO4 at 0 h postchallenge (26%) was significantly less than the positive control (77%). The mortalities of challenged fish treated at 1, 2 or 4 h postchallenge were not significantly different from the positive control fish. The results suggest that KMnO4 has a clear therapeutic value in early stages of columnaris infection but limited therapeutic value once the infection has progressed.

This range-finding study determined the optimum concentration of copper sulfate (CuSO4) for fungus control on eggs of channel catfish Ictalurus punctatus. The study consisted of five CuSO4 concentrations (2.5, 5, 10, 20, and 40 mg/L) and an untreated control in a flow-through system. A single spawn was used for each replication (N=4). Eggs were treated daily until the embryos reached the eyed stage. When hatching was complete for all viable eggs, fry were counted to determine the percent survival in each treatment. Fungal growth was severe in the untreated controls; survival of hatched fry in the control group was approximately 2%. The optimum CuSO4 treatment, as determined by percent survival of hatched fry, was 10 mg/L daily (69% survival); survival for this treatment group was significantly different from that for the controls. Very little fungus was present in treatments receiving 10-mg/L CuSO4 or higher except in one replication that had approximately 40% unfertilized eggs. The average survival rates in the 0-, 2.5-, 5-, 10-, 20-, and 40-mg/L CuSO4 treatments were 2, 34, 50, 69, 59, and 51%, respectively.

International audience; Cerebral malaria (CM) is characterized by accumulation of circulating cells within brain microvessels, among which platelets play an important role. In vitro, platelets modulate the cytoadherence of Plasmodium falciparum-parasitized red blood cells (PRBCs) to brain endothelial cells. Here we show for the first time that platelet microparticles (PMPs) are able to bind to PRBCs, thereby transferring platelet antigens to the PRBC surface. This binding is largely specific to PRBCs, because PMPs show little adherence to normal red blood cells. PMP adherence is also dependent on the P. falciparum erythrocyte membrane protein 1 variant expressed by PRBCs. PMP binding to PRBCs decreases after neutralization of PRBC surface proteins by trypsin or after treatment of PMPs with a mAb to platelet-endothelial cell adhesion molecule-1 (CD31) and glycoprotein IV (CD36). Furthermore, PMP uptake is a dynamic process that can be achieved by human brain endothelial cells (HBECs), inducing changes in the endothelial phenotype. Lastly, PMPs dramatically increase PRBC cytoadherence to HBECs. In conclusion, our study identifies several mechanisms by which PMPs may participate in CM pathogenesis while interacting with both PRBCs and HBECs. PMPs thereby provide a novel target for antagonizing interactions between vascular cells that promote microvascular sludging and blood brain barrier alteration during CM.

1 Harry K. Dupree Stuttgart National Aquaculture Research Center, U. S. Department of Agriculture, AgriculturalResearch Service, Stuttgart, AR, USA2 The University of Southern Mississippi, Department of Costal Sciences, Gulf Coast Research Laboratory, OceanSprings, MS, USA3 Aquaculture/Fisheries Center, Department of Aquaculture/Fisheries, University of Arkansas at Pine Bluff, AR, USA

A trial was performed to evaluate the efficacy of diquat (6,7-dihydrodipyrido[1,2-a:2',1'-c]pyrazinediium dibromide) against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus. Diquat is an Environmental Protection Agency-approved herbicide and has the potential to be legally and practically used against columnaris. Channel catfish were challenged, by cutaneous abrasion, and waterborne exposure to F. columnare and treated once at 22-h post-challenge with 2.5, 5.0, 10.0 and 15 mg L(-1) of diquat active ingredient for 6 h. At the conclusion of the trial, 21-day post-challenge, diquat at 5.0, 10.0 and 15 mg L(-1) significantly (P0.05) reduced the mortality of infected fish from 95% in the challenged non-treated fish to 68%, 59% and 49%, respectively. In vitro, the minimum inhibitory concentration (MIC) of 23 isolates of F. columnare was assayed. The majority of the isolates had an MIC value of 5 microg mL(-1) (15 of the 23 isolates). Infected fish exhibited acute clinical signs similar to a natural infection. The skin had severe ulcerative necrotizing dermatitis and the muscles had severe necrotizing myositis. The gills had severe multifocal necrotizing branchitis. The results demonstrate that diquat would reduce mortalities caused by an acute columnaris infection.

An experiment was performed to evaluate the efficacy of potassium permanganate (KMnO4) as a prophylactic and therapeutic treatment of an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus. Fish were cutaneously abraded and divided into five treatment groups: (i) challenged by waterborne exposure to F. columnare and not treated with KMnO4 (positive control), (ii) challenged and simultaneously treated with KMnO4, (iii) challenged and treated with KMnO4 at 1, 6 and 9 days post-challenge, (iv) not challenged and treated with KMnO4 at 1, 6 and 9 days post-challenge (first negative control) and (v) not challenged and not treated (second negative control). The dosing of KMnO4 was 2.0 mg L(-1) above the potassium permanganate demand for 2 h duration. The survival of the group challenged and simultaneously treated with KMnO4 (99%) was significantly higher than the positive control (78%) and was not significantly different from the negative control groups. The challenged fish treated with KMnO4 post-challenge had 7% higher survival than the positive control (85% compared with 78%), but that difference was not statistically significant. The results demonstrate that KMnO4 has a clear prophylactic value but probably a marginal therapeutic value once the infection has established.

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. Using murine models of malaria, we found much greater up-regulation of a number of chemokine mRNAs, including those for CXCR3 and its ligands, in the brain during fatal murine CM (FMCM) than in a model of non-CM. Expression of CXCL9 and CXCL10 RNA was localized predominantly to the cerebral microvessels and in adjacent glial cells, while expression of CCL5 was restricted mainly to infiltrating lymphocytes. The majority of mice deficient in CXCR3 were found to be protected from FMCM, and this protection was associated with a reduction in the number of CD8+ T cells in brain vessels as well as reduced expression of perforin and FasL mRNA. Adoptive transfer of CD8+ cells from C57BL/6 mice with FMCM abrogated this protection in CXCR3−/− mice. Moreover, there were decreased mRNA levels for the proinflammatory cytokines IFN-γ and lymphotoxin-α in the brains of mice protected from FMCM. These data suggest a role for CXCR3 in the pathogenesis of FMCM through the recruitment and activation of pathogenic CD8+ T cells.

Channel catfish, Ictalurus punctatus (Rafinesque), with or without a preliminary 24 h exposure to 2 mg copper sulphate L(-1), were challenged with 7.5 x 10(6) colony forming units L(-1) of Edwardsiella ictaluri to determine the effect of copper sulphate on disease resistance. Catfish previously exposed to copper sulphate were significantly more resistant to the bacterial challenge than those not exposed. Catfish not exposed to copper sulphate suffered 35.5% mortality while catfish exposed to copper sulphate experienced 14.1% mortality. Copper concentrations were the same in tank waters of both exposed and control fish at the time of challenge, eliminating the possibility that copper in the water may have been toxic to bacteria. Copper concentrations in freeze dried and ground tissues of unexposed, exposed, and purged channel catfish were highest in fish before copper sulphate exposures suggesting that elevated tissue levels of copper were not responsible for the increased resistance to bacterial challenge. Competition for sites of bacterial attachment to gill or epithelial cells may account for the reduction in mortality; although this is not supported by the low copper content of fish tissue after copper exposure.

Phagocyte-derived reactive oxygen species have been implicated in the clearance of malaria infections. We investigated the progression of five different strains of murine malaria in gp91phox−/−mice, which lack a functional NADPH oxidase and thus the ability to produce phagocyte-derived reactive oxygen species. We found that the absence of functional NADPH oxidase in the gene knockout mice had no effect on the parasitemia or total parasite burden in mice infected with either resolving (Plasmodium yoeliiandPlasmodium chabaudiK562) or fatal (Plasmodium bergheiANKA,Plasmodium bergheiK173 andPlasmodium vinckei vinckei) strains of malaria. This lack of effect was apparent in both primary and secondary infections withP. yoeliiandP. chabaudi. There was also no difference in the presentation of clinical or pathological signs between the gp91phox−/−or wild-type strains of mice infected with malaria. Progression ofP. bergheiANKA andP. bergheiK173 infections was unchanged in glutathione peroxidase-1 gene knockout mice compared to their wild-type counterparts. The rates of parasitemia progression in gp91phox−/−mice and wild-type mice were not significantly different when they were treated withl-NG-methylarginine, an inhibitor of nitric oxide synthase. These results suggest that phagocyte-derived reactive oxygen species are not crucial for the clearance of malaria parasites, at least in murine models.

Cerebral malaria (CM) kills more than 1 million children each year. Using a murine model of CM, we investigated the role of Fas-Fas ligand interactions in the neuropathogenesis of this disease. Lpr and Gld mice, deficient in Fas and Fas ligand, respectively, were protected from fatal CM, although they demonstrated some pathological features associated with CM in the wild type mouse. Fas-Fas ligand mRNA and protein expression were increased in the brain in mice with CM, and activated caspase-3-positive apoptotic astrocytes were observed. We suggest that Fas-mediated apoptosis of astrocytes is likely to be a critical factor in late-stage murine CM pathogenesis.

Cerebral malaria is a serious complication of Plasmodium falciparum infection. We have investigated the role of perforin in the pathogenesis of cerebral malaria in a murine model (Plasmodium berghei ANKA (PbA) infection). C57BL/6 mice demonstrated the typical neuropathological symptoms of experimental cerebral malaria infection from day 5p.i. and became moribund on day 6p.i. This pathology was not seen in PbA-infected, perforin-deficient (pfp-/-) mice. From days 5-6p.i. onwards there was a significant increase in mRNA for granzyme B and CD8, but not CD4, in brain tissue from PbA-infected C57BL/6 and pfp-/- mouse brains. Perforin mRNA was strongly increased in the brains of PbA-infected C57BL/6 mice on day 6p.i. Immunohistochemistry revealed increased perforin staining and elevated numbers of CD8(+) cells within the cerebral microvessels in PbA-infected C57BL/6 at days 5 and 6p.i. compared with uninfected animals. At day 6p.i., there were TUNEL-positive cells and activated caspase-3 positive cells of endothelial morphology in the CNS of PbA-infected C57BL/6 mice. The TUNEL-positive cells were greatly reduced in pfp-/- mice. These results suggest that CD8(+)T lymphocytes induce apoptosis of endothelial cells via a perforin-dependent process, contributing to the fatal pathogenic process in murine cerebral malaria.

Production of reactive oxygen species by redox cycling in the presence of low levels of oxygen has been studied as a possible approach to anti-protozoal chemotherapeutic strategy. Incubation of the diplomonad flagellateGiardia intestinaliswith 2-methy-1,4-naphthoquinone (menadione), under anaerobic conditions, gave UV absorption changes characteristic of reduction to menadiol; partial reversal was observed on admitting O2. Under microaerobic conditions, similar to those on the surface of the jejunal mucosa, trophozoites consumed O2rapidly in the presence of menadione; reaction products included singlet O2(monitored by single photon counting of O2-dependent low-level chemiluminescence) and H2O2(measured by the formation of Complex I of microperoxidase). Trophozoites became swollen and incapable of regulatory volume control; these irreversible responses led to loss of motility, cessation of flagellar activity and cell death. Comparison of the sensitivities of trophozoites to metronidazole and menadione gave LC50values (μg ml−1) of 1·2 and 0·7, respectively; corresponding values for cysts (measured byin vitroexcystation capacities) were >50 and 1·3. Menadione (LD50in mice, 0·5 g kg−1) is therefore a potentially more useful and general anti-giardial agent than metronidazole, as it is active against cysts as well as trophozoites.

This paper derives the Vapnik Chervonenkis dimension of several natural subclasses of pattern languages. For classes with unbounded VC-dimension, an attempt is made to quantify the “rate of growth” of VC-dimension for these classes. This is achieved by computing, for each n, size of the “smallest” witness set of n elements that is shattered by the class. The paper considers both erasing (empty substitutions allowed) and nonerasing (empty substitutions not allowed) pattern languages. For erasing pattern languages, optimal bounds for this size — within polynomial order — are derived for the case of 1 variable occurrence and unary alphabet, for the case where the number of variable occurrences is bounded by a constant, and the general case of all pattern languages. The extent to which these results hold for nonerasing pattern languages is also investigated. Some results that shed light on efficient learning of subclasses of pattern languages are also given.

Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons. Proteolysis has long been suspected to regulate extracellular SP concentrations but data in support of this hypothesis is scarce. Here, we provide evidence that proteolysis controls SP levels in the spinal cord. Using peptidomics to detect and quantify endogenous SP fragments, we identify the primary SP cleavage site as the C-terminal side of the ninth residue of SP. If blocking this pathway increases SP levels, then proteolysis controls SP concentration. We performed a targeted chemical screen using spinal cord lysates as a proxy for the endogenous metabolic environment and identified GM6001 (galardin, ilomastat) as a potent inhibitor of the SP(1-9)-producing activity present in the tissue. Administration of GM6001 to mice results in a greater-than-three-fold increase in the spinal cord levels of SP, which validates the hypothesis that proteolysis controls physiological SP levels.

We have established a novel in vitro co-culture system of human brain endothelial cells (HBEC), Plasmodium falciparum parasitised red blood cells (iRBC) and peripheral blood mononuclear cells (PBMC), in order to simulate the chief pathophysiological lesion in cerebral malaria (CM). This approach has revealed a previously unsuspected pro-inflammatory role of the endothelial cell through potentiating the production of interferon (IFN)-γ by PBMC and concurrent reduction of interleukin (IL)-10. The IFN-γ increased the expression of CXCL10 and intercellular adhesion molecule (ICAM)-1, both of which have been shown to be crucial in the pathogenesis of CM. There was a shift in the ratio of IL-10:IFN-γ protein from >1 to

Although its etiology remains unknown, evidence has accumulated to support an autoimmune pathogenesis for alopecia areata. Our review summarizes the immunologic data and also examines the role of genetics, atopy, and psychologic stress in this disorder. Until etiology is better understood, treatments for alopecia areata are likely to remain palliative. Nevertheless, newer therapies such as photochemotherapy, topical immunotherapy, and perhaps systemic immunotherapy (e.g., inosiplex) offer new hope for patients with extensive disease.

It has been shown previously that alopecia areata can be treated with dinitrochlorobenzene (DNCB) and other contact allergens. Whether these agents work by inducing immunologic stimulation or simply a nonspecific inflammatory reaction has not been definitively demonstrated. To test the relative importance of these two mechanisms, we have randomly studied 22 patients with alopecia areata to whom either DNCB or croton oil was applied topically. Sixty-three percent of patients without spontaneous regrowth of hair regrew hair after DNCB application. None of those treated with croton oil regrew hair when treated later with DNCB. Therefore, a proved contact allergen was shown to be required for therapeutic success. Patient acceptance of the induced contact dermatitis was excellent. In light of recent data on the mutagenicity of DNCB to bacteria, other contact allergens for topical immunotherapy are being sought.

A massive infection of metacercariae, particularly of the white grub (Posthodiplostomum m. minimum), was found in fathead minnows (Pimephales promelas) taken in November, 1979 from a commercial baitfish farm in Missouri. More than 2000 metacercariae were found in one fish 6 cm long. Low-grade mortality was occurring, and histologic sections showed that an intensive inflammatory response had resulted from the infection. The body cavity also contained a large volume of ascitic fluid with many blood cells, of which 92% were leucocytes. Moderate numbers of metacercariae caused a lesser inflammatory response.

Twenty-two patients with alopecia areata were treated with a combination of topical 0.1% 8-methoxypsoralen and UVA (PUVA). Eight of the twenty-two patients (36.3%) responded with excellent regrowth (terminal hair in at least 75% of the treated scalp), and two patients (9.1%) showed good regrowth (terminal hair in 50% to 75% of the treated scalp). The mean total UVA exposure and the mean total number of treatments for the entire treatment course for these responders was 171.7 joules/cm2 and 47.4 treatments, respectively. Eight of the nine responders available for follow-up experienced some degree of relapse when PUVA treatments were tapered or during a follow-up period (mean, 8.3 months) after treatment was discontinued. Despite the failure of topical PUVA to change the long-term course of alopecia, the combination of PUVA with other therapeutic modalities may result in the prolongation of the beneficial effect in selected patients. The mechanism of action of PUVA in alopecia areata might involve an immunomodulatory effect.

An 8-year-old girl initially manifested clinical and histopathologic findings of circumscribed scleroderma. She had both linear and plaque lesions. Although she was free of constitutional symptoms, laboratory evaluation revealed substantial evidence of systemic lupus erythematosus (SLE). This included a positive anti-nuclear antibody test (rim pattern), positive LE cell preparation test, elevated anti-double-stranded DNA activity, positive lupus band test (IgM and C3) in involved and uninvolved skin, and renal biopsy findings consistent with SLE. Sclerodermatous change as an initial sign of SLE is rare. We review previous reports of an association of circumscribed scleroderma, especially the linear form, with serologic evidence of systemic autoimmune disease.

Forty-five children with alopecia areata were prospectively studied by means of both clinical and laboratory evaluation for evidence of endocrine diseases and autoantibodies. Twenty-four percent had an abnormality as determined by one or more thyroid function studies (thyroxine, triiodothyronine, and thyroid-stimulating hormone) and/or elevation of microsomal antibody levels. In 16%, smooth muscle antibody was present, and in 4%, parietal cell antibody was present. Routine thyroid function testing is recommended for all children with alopecia areata.

A patient presented with a solitary plaque initially interpreted on light microscopy as benign lymphoplasia. Monoclonal antibody studies were used to define the B cell origin of the infiltrate and to characterize its probable malignant potential. Monoclonal antibody typing is a useful technique to evaluate lymphocytic infiltrates that may be difficult to categorize on the basis of routine light microscopy.

Confluent and reticulated papillomatosis of Cougerot and Carteaud (CRP) is a distinctive clinicopathologic entity, of unknown etiology, which shows some resemblance to pityriasis versicolor. A case of CRP that showed a signific ant therapeutic response to topical selenium sulfide is reported. In previously described cases, response to this agent and to other medications, both antifungal and keratolytic, has been variable. A review of these cases reveals conflicting evidence regarding the questionable role of Malassezia furfur in CRP.

• Epidermodysplasia verruciformis is a rare, often hereditary disease characterized by a generalized cutaneous infection with human papillomavirus (HPV), depressed cell-mediated immunity, and a propensity for transformation of the warty lesions to squamous cell carcinoma on primarily sun-exposed areas of the skin. A 37-year-old man with congenital lymphatic dysplasia and a history of squamous cell carcinoma of the groin and foot was observed by us to have edema of all four extremities, numerous flat warts, and pityriasis versicolor-like papules over the trunk and arms. Condylomatous lesions were noted in the groin and a periungual verrucous nodule on the thumb. Biopsies showed the trunk and arm lesions to be verrucae and the thumb lesion to be Bowen's disease. Results of molecular hybridization studies from four lesions of the arms showed the presence of only HPV 3 DNA; HPV 16-related DNA was detected in the intraepidermal carcinoma on the thumb. Immunologic evaluation revealed anergy to routine skin testing, depressed mitogen-stimulated lymphocyte transformation, decreased B-lymphocyte count, and a severe reversal of the T-lymphocyte helper:suppressor ratio. (Arch Dermatol1987;123:1511-1516)

A 27-year-old woman developed a chronic, recurrent eruption of the face and upper extremities with the clinical and histopathologic features of recurrent granulomatous dermatitis with eosinophilia (Wells'syndrome). As described in 15 previously reported cases, this disorder is characterized by two clinical phases (ensinophilic cellulitis and granuloniatou plaque phase) and three histopathologic stages. The latter are particularly remarkable for a diffuse dermal and subcutaneous eosinophilia in acute lesions and scattered flame figures in chronic lesions. Distinctive findings in this case were the predominance of facial involvement and the symptomatic response to topical corticosteroids. Although etiology and pathogenesis are unknown, we feel that Wells'syndrome is a unique yet rarely recognized clinicopathologic entity.

Alternaria species are common plant pathogens, but a rare cause of human infection. Previously reported cases of cutaneous alternariosis (Alternaria alternata) in both healthy and immunosuppressed hosts have been characterized by a chronic localized ulcerative eruption in exposed sites. Dermal granulomatous infiltration and variable epidermal changes, with hyphae in both the dermis and epidermis, are characteristic. We present a case with certain unique features. This is the first reported human infection with Alternaria dianthicola. In addition, our patient showed the unusual presentation of clinically noninflammatory subcutaneous nodules of the chest wall. The subcutaneous location of the granulomatous process may have been attributable to deep traumatic inoculation. The circumscribed nodular quality of the infection allowed a surgical therapeutic approach.

Amphileptus branchiarum is a protozoan parasite of fish in North America, Europe and Asia. In North America (Arkansas) it usually occurs on the gills of cyprinids from July to October. It seldom causes disease epizootics but may be responsible for epithelial hyperplasia and cell displacement in branchial tissue.

The efficacy of the topical allergen squaric acid dibutylester (SADBE) was investigated in the treatment of twenty-six patients with alopecia areata or alopecia totalis. The patients had their disease for a mean duration of 8.3 years (range, 3 months to 27 years). Sensitization was attempted with 2% SADBE in acetone. Five individuals could not be sensitized. The twenty-one sensitized patients were treated with topical applications of 0.001% to 1.0% SADBE in acetone adjusted to produce and maintain dermatitis for an average of 21 weeks. Eleven (52%) had excellent responses consisting of complete regrowth in six and cosmetically acceptable regrowth in five. The average treatment time for regrowth was 11 weeks (range, 5-20 weeks). Ten (48%) had no clinical response after an average of 19 weeks of therapy (range, 4-42 weeks). Topical SADBE is an effective therapy for some patients with long-standing alopecia areata.

Despite the best efforts of public health authorities, malaria remains a major global health concern, with estimates ranging from 300 to 500 million people infected every year (10, 48). The majority of deaths attributed to malaria occur in sub-Saharan Africa as a result of infection by Plasmodium falciparum. One of the major life-threatening complications of P. falciparum infection is cerebral malaria (CM), which is characterized by convulsions and unarousable coma. Although only a small percentage of infected individuals develop CM, the mortality rate once it has developed is around 20% (47). While there are competing hypotheses about the etiology of CM, there is strong evidence that it is an immunopathological process (9, 26). In particular, animal studies using the rodent malaria strain Plasmodium berghei ANKA have been revealing. For P. berghei ANKA infections of susceptible mouse strains, the development of CM is dependent upon a variety of immunological processes. For example, the production during the course of infection of cytokines such as gamma interferon (IFN-γ) (21, 39), as well as the presence of both CD4+ and CD8+ T cells (4, 24, 35, 49), has been shown to be essential for CM pathology to occur. Investigations of CM pathogenesis have examined almost exclusively the immune processes immediately preceding the development of cerebral manifestations, that is, the end-stage pathological processes. Thus, there is little understanding of the very early immune system responses to malarial parasites. In many infection models, such early innate immune processes have been shown to critically influence the later development of adaptive immune responses (reviewed in references 27 and 37), so it seems possible that early responses may also influence the immunopathology of CM. We therefore investigated the early immune responses to two closely related Plasmodium strains and correlated these with the pathological outcome. Susceptible mice infected with Plasmodium berghei ANKA succumb to CM 6 to 7 days after infection. In contrast, mice infected with Plasmodium berghei K173 develop high parasitemia levels and low hematocrits and die without cerebral symptoms 2 to 3 weeks after infection. We found that 24 h after parasite inoculation with P. berghei K173, but not P. berghei ANKA, there was a transient production of a range of cytokines in the spleen, most notably of IFN-γ. Upon simultaneous infection of mice with both P. berghei ANKA and P. berghei K173, a similar pattern of cytokine production to that seen with K173 alone was seen, and this correlated with the absence of later CM. This suggested that an active suppression of immunopathological processes was occurring in P. berghei K173 infection and that this dominated over the processes usually occurring in P. berghei ANKA infection. Since IFN-γ may be involved in immunosuppression through the induction of indoleamine 2,3-dioxygenase (IDO), the cellular processes occurring during early IFN-γ production were investigated for P. berghei K173 infection, and production of the cytokine was found to be β2-microglobulin (β2-M) dependent.