BACKGROUND

HIV-associated tuberculosis is difficult to diagnose and results in high mortality. Frequent extra-pulmonary presentation, inability to obtain sputum, and paucibacillary samples limits the usefulness of nucleic-acid amplification tests and smear microscopy. We therefore assessed a urine-based, lateral flow, point-of-care, lipoarabinomannan assay (LAM) and the effect of a LAM-guided anti-tuberculosis treatment initiation strategy on mortality.

METHODS

We did a pragmatic, randomised, parallel-group, multicentre trial in ten hospitals in Africa--four in South Africa, two in Tanzania, two in Zambia, and two in Zimbabwe. Eligible patients were HIV-positive adults aged at least 18 years with at least one of the following symptoms of tuberculosis (fever, cough, night sweats, or self-reported weightloss) and illness severity necessitating admission to hospital. Exclusion criteria included receipt of any anti-tuberculosis medicine in the 60 days before enrolment. We randomly assigned patients (1:1) to either LAM plus routine diagnostic tests for tuberculosis (smear microscopy, Xpert-MTB/RIF, and culture; LAM group) or routine diagnostic tests alone (no LAM group) using computer-generated allocation lists in blocks of ten. All patients were asked to provide a urine sample of at least 30 mL at enrolment, and trained research nurses did the LAM test in patients allocated to this group using the Alere Determine tuberculosis LAM Ag lateral flow strip test (Alere, USA) at the bedside on enrolment. On the basis of a positive test result, the nurses made a recommendation for initiating anti-tuberculosis treatment. The attending physician made an independent decision about whether to start treatment or not. Neither patients nor health-care workers were masked to group allocation and test results. The primary endpoint was 8-week all-cause mortality assessed in the modified intention-to-treat population (those who received their allocated intervention). This trial is registered with ClinicalTrials.gov, number NCT01770730.

RESULTS

Between Jan 1, 2013, and Oct 2, 2014, we screened 8728 patients and randomly assigned 2659 to treatment (1336 to LAM, 1323 to no LAM). 108 patients did not receive their allocated treatment, mainly because they did not meet the inclusion criteria, and 23 were excluded from analysis, leaving 2528 in the final modified intention-to-treat analysis (1257 in the LAM group, 1271 in the no LAM group). Overall all-cause 8-week mortality occurred in 578 (23%) patients, 261 (21%) in LAM and 317 (25%) in no LAM, an absolute reduction of 4% (95% CI 1-7). The risk ratio adjusted for country was 0·83 (95% CI 0·73-0·96), p=0·012, with a relative risk reduction of 17% (95% CI 4-28). With the time-to-event analysis, there were 159 deaths per 100 person-years in LAM and 196 per 100 person-years in no LAM (hazard ratio adjusted for country 0·82 [95% CI 0·70-0·96], p=0·015). No adverse events were associated with LAM testing.

CONCLUSIONS

Bedside LAM-guided initiation of anti-tuberculosis treatment in HIV-positive hospital inpatients with suspected tuberculosis was associated with reduced 8-week mortality. The implementation of LAM testing is likely to offer the greatest benefit in hospitals where diagnostic resources are most scarce and where patients present with severe illness, advanced immunosuppression, and an inability to self-expectorate sputum.

BACKGROUND

European Developing Clinical Trials Partnership, the South African Medical Research Council, and the South African National Research Foundation.

BACKGROUND

Mammalian Toll-like receptor (TLR) proteins are pattern recognition receptors for a diverse array of bacterial and viral products. Gram negative bacterial lipopolysaccharide (LPS) activates cells through TLR4, whereas the mycobacterial cell wall glycolipids, lipoarabinomannan (LAM) and mannosylated phosphatidylinositol (PIM), activate cells through TLR2. Furthermore, short term culture filtrates of M. tuberculosis bacilli contain a TLR2 agonist activity, termed soluble tuberculosis factor (STF), that appears to be PIM. It was recently shown that stimulation of RAW264.7 murine macrophages by LPS, LAM, STF, and PIM rapidly activated NF-kappaB, AP1, and MAP kinases.

RESULTS

This study shows that signalling by TLR2 and TLR4 also activates the protein kinase Akt, a downstream target of phosphatidylinositol-3'-kinase (PI-3-K). This finding suggests that activation of PI-3-K represents an additional signalling pathway induced by engagement of TLR2 and TLR4. Subsequently, the functional responses induced by the different TLR agonists were compared. LPS, the mycobacterial glycolipids, and the OspC lipoprotein (a TLR2 agonist) all induced macrophages to secrete tumour necrosis factor alpha (TNFalpha), whereas only LPS could induce nitric oxide (NO) secretion. Human alveolar macrophages also exhibited a distinct pattern of cellular response after stimulation with TLR2 and TLR4 agonists. Specifically, LPS induced TNFalpha, MIP-1beta, and RANTES production in these cells, whereas the TLR2 agonists induced only MIP-1beta production.

CONCLUSIONS

Together, these data show that different TLR proteins mediate the activation of distinct cellular responses, despite their shared ability to activate NF-kappaB, AP1, MAP kinases, and PI-3-K.

Macrophages (MAC) and polymorphonuclear granulocytes (PNG) are professional phagocytes which perform essential functions in antibacterial defense. The intracellular bacterium Mycobacterium tuberculosis persists and replicates in resting macrophages. Although it is generally assumed that activated MAC are central to protection against M. tuberculosis, PNG may also contribute to defense. We wondered whether PNG produce proinflammatory chemokines after stimulation by M. tuberculosis or its major cell wall component, lipoarabinomannan (LAM). In this study, we showed that M. tuberculosis- and LAM-activated human PNG secrete the leukocyte attractant interleukin-8 (IL-8) and the PNG-specific chemokine GRO-alpha in a dose-dependent manner. Treatment of PNG with the leukotriene-B4 inhibitor MK-886 prior to stimulation with M. tuberculosis or LAM partially blocked IL-8 and GRO-alpha induction, suggesting involvement of the 5-lipoxygenase pathway in the secretion of these chemokines. We conclude that PNG contribute to early resistance to M. tuberculosis via chemokine secretion.

Lipoarabinomannans (LAMs) are glycolipids from the mycobacterial cell wall that exhibit various biological activities, including proinflammatory and anti-inflammatory responses. However, little is known about the properties of lipomannans (LMs), considered to be precursors of LAMs. In this study, we provide evidence that LMs purified from Mycobacterium chelonae and a clinical strain of Mycobacterium kansasii stimulated mRNA expression and secretion of TNF-alpha and IL-8 from human macrophage-like differentiated THP-1 cells. In contrast to LMs, LAMs were not able to induce a significant cytokine-inducing effect. The mechanism of activation by LMs was investigated using various Abs raised against surface receptors for multiple bacterial products. The presence of anti-CD14 or anti-Toll-like receptor 2 (TLR2) Abs profoundly affected production of TNF-alpha and IL-8, suggesting that both CD14 and TLR2 participate in the LM-mediated activation process. Furthermore, stimulation of cells was dependent on the presence of the LPS-binding protein, a plasma protein that transfers glycolipids to CD14. Chemical degradation of the arabinan domain of mannose-capped LAM from M. kansasii, which presented no cytokine-eliciting effect, restored the cytokine-inducing activity at a level similar to those of LMs. These results support the hypothesis that the presence of an arabinan in LAMs prevents the interaction of these glycolipids with TLR2/CD14 receptors. In addition, we found that phosphatidylinositol dimannosides isolated from M. kansasii did not induce cytokine secretion. This study suggests that LMs isolated from different mycobacterial species participate in the immunomodulation of the infected host and that the D-mannan core of this glycolipid is essential for this function.

Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.

Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a "humanized" dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1-3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (<1/1,000,000) at most times over 2 years. In one patient, a reappearance of PBMC containing the huM10 gene, but not the FX gene, occurred concomitant with a rise in the HIV-1 viral load during a period of nonadherence to the antiretroviral regimen. Unique clones of gene-marked PBMC were detected by LAM-PCR during the time of elevated HIV-1 levels. These findings indicate that there was a selective survival advantage for PBMC containing the huM10 gene during the time of increased HIV-1 load.

Lymphangioleiomyomatosis (LAM) affects exclusively women of reproductive age, involves the lungs and axial lymphatic system, and is frequently complicated with renal angiomyolipomas. LAM lesions are generated by the proliferation of LAM cells with mutations of one of the tuberous sclerosis complex (TSC) genes. Recent studies indicate that LAM cells can migrate or metastasize to form new lesions in multiple organs, although they show a morphologically benign appearance. In the previous study, we reported LAM-associated lymphangiogenesis and implicated its role in the progression of LAM. In this study, we further focused on the lymphatic abnormalities in LAM: LAM-associated chylous fluid (5 pleural effusion and 2 ascites), surgically resected diaphragm (1 patient), and axial lymphatic system including the thoracic duct, lymph nodes at various regions, and diaphragmatic lymphatic system (5 autopsy cases). We demonstrated that LAM cell clusters enveloped by lymphatic endothelial cells (LCC) in all chylous fluid examined. We identified LAM lesion in the diaphragm (2 of 5 autopy cases and one surgical specimen), thoracic duct (5 of 5), and lymph nodes (retroperitoneal (5 of 5), mediastinal (4 of 5), left venous angle (5 of 5) with total positive rate of 68% to 88% at each region of the lymph node, but less frequent or none at remote lymph nodes located away from the axial lymph trunk (cervical [1 of 5] and axillary [0 of 5]). LCCs were identified in intra-LAM lesional lymphatic channels where LAM cells proliferate along lymphatic system. In in vitro culture system, LCC can fragment into each proliferating LAM cell. These findings suggest that LAM-associated lymphangiogenesis demarcates LAM lesion into bundle- or fascicle-like structure and eventually shed LCC into the lymphatic circulation and that LCCs play a central role in the dissemination of LAM lesion.

Two classes of enzymatic mechanisms that proceed by free radical chemistry initiated by the 5'-deoxyadenosyl radical are discussed. In the first class, the mechanism of the interconversion of L-lysine and L-beta-lysine catalyzed by lysine 2,3-aminomutase (LAM) involves four radicals, three of which have been spectroscopically characterized. The reversible formation of the 5'-deoxyadenosyl radical takes place by the chemical cleavage of S-adenosylmethionine (SAM) reacting with the [4Fe-4S]+ center in LAM. In other reactions of SAM with iron-sulfur proteins, SAM is irreversibly consumed to generate the 5'-deoxyadenosyl radical, which activates an enzyme by abstracting a hydrogen atom from an enzymatic glycyl residue to form a glycyl radical. The glycyl radical enzymes include pyruvate formate-lyase, anaerobic ribonucleotide reductase from Escherichia coli, and benzylsuccinate synthase. Biotin synthase and lipoate synthase are SAM-dependent [4Fe-4S] proteins that catalyze the insertion of sulfur into unactivated C-H bonds, which are cleaved by the 5'-deoxyadenosyl radical from SAM. In the second class of enzymatic mechanisms using free radicals, adenosylcobalamin-dependent reactions, the 5'-deoxyadenosyl radical arises from homolytic cleavage of the cobalt-carbon bond, and it initiates radical reactions by abstracting hydrogen atoms from substrates. Three examples are described of suicide inactivation through the formation of exceptionally stable free radicals at enzymatic active sites.

Spinal cord injury (SCI) causes motor and sensory deficits that impair functional performance. While more functional recovery occurs with greater white matter sparing (WMS), it is unclear which locomotor features are more vulnerable to SCI than others, if recovery of certain features depends on specific amounts of WMS, and whether motor recovery patterns differ from sensory recovery. Locomotor and sensory recovery after graded contusive SCI with cord displacements of 0.3, 0.5, 0.7, 0.9, 1.1, 1.25, and 1.3 mm was examined for 6 weeks in 80 female Sprague-Dawley rats. Seven SCI gradations resulted in three locomotor performance levels measured with BBB (P < 0.01): High: laminectomy (LAM) controls and 0.3 (19.87 +/- 0.35 SEM); Intermediate: 0.5-0.9 (13.71 +/- 0.32); and Low: 1.1-1.3 (9.23 +/- 0.36). Normal paw position was most susceptible to SCI requiring 90% WMS, while consistent plantar stepping was least susceptible depending on 10% WMS. A threshold at the 0.9 severity for coordination, toe clearance, and nearly normal trunk stability and tail usage required 25% WMS. Analysis of interlimb coordination using new phase dispersion (PD) techniques delineated three recovery patterns: synchronous (0.3), modified concordance (0.5, 0.7), and disengaged (0.9, 1.1). Lesion severity correlated to WMS (r(2) = 0.96) and to BBB (r(2) = 0.87) by nonlinear polynomial regressions. Mechanical allodynia developed only after injuries resulting in < or =10% WMS. Nonlinear motor and sensory recovery patterns suggest that small reparative changes may substantially improve function in individuals with SCI. A hierarchical locomotor recovery based on simple segmental versus complex supraspinal motor control is proposed.

Root-deposited photosynthate (rhizodeposition) is an important source of readily available carbon (C) for microbes in the vicinity of growing roots. Plant nutrient availability is controlled, to a large extent, by the cycling of this and other organic materials through the soil microbial community. Currently, our understanding of microbial community dynamics associated with rhizodeposition is limited. We used a (13)C pulse-chase labeling procedure to examine the incorporation of rhizodeposition into individual phospholipid fatty acids (PLFAs) in the bulk and rhizosphere soils of greenhouse-grown annual ryegrass (Lolium multiflorum Lam. var. Gulf). Labeling took place during a growth stage in transition between active root growth and rapid shoot growth on one set of plants (labeling period 1) and 9 days later during the rapid shoot growth stage on another set of plants (labeling period 2). Temporal differences in microbial community composition were more apparent than spatial differences, with a greater relative abundance of PLFAs from gram-positive organisms (i15:0 and a15:0) in the second labeling period. Although more abundant, gram-positive organisms appeared to be less actively utilizing rhizodeposited C in labeling period 2 than in labeling period 1. Gram-negative bacteria associated with the 16:1omega5 PLFA were more active in utilizing (13)C-labeled rhizodeposits in the second labeling period than in the first labeling period. In both labeling periods, however, the fungal PLFA 18:2omega6,9 was the most highly labeled. These results demonstrate the effectiveness of using (13)C labeling and PLFA analysis to examine the microbial dynamics associated with rhizosphere C cycling by focusing on the members actively involved.

OBJECTIVE

Endoscopic transmural drainage/debridement of pancreatic walled-off necrosis (WON) has been performed using double-pigtail plastic (DP), fully covered self-expanding metal stents (FCSEMSs), or the novel lumen-apposing fully covered self-expanding metal stent (LAMS). Our aim was to perform a retrospective cohort study to compare the clinical outcomes and adverse events of EUS-guided drainage/debridement of WON with DP stents, FCSEMSs, and LAMSs.

METHODS

Consecutive patients in 2 centers with WON managed by EUS-guided debridement were divided into 3 groups: (1) those who underwent debridement using DP stents, (2) debridement using FCSEMSs, (3) debridement using LAMSs. Technical success (ability to access and drain a WON by placement of transmural stents), early adverse events, number of procedures performed per patient to achieve WON resolution, and long-term success (complete resolution of the WON without need for further reintervention at 6 months after treatment) were evaluated.

RESULTS

From 2010 to 2015, 313 patients (23.3% female; mean age, 53 years) underwent WON debridement, including 106 who were drained using DP stents, 121 using FCSEMSs, and 86 using LAMSs. The 3 groups were matched for age, cause of the pancreatitis, WON size, and location. The cause of the patients' pancreatitis was gallstones (40.6%), alcohol (30.7%), idiopathic (13.1%), and other causes (15.6%). The mean cyst size was 102 mm (range, 20-510 mm). The mean number of endoscopy sessions was 2.5 (range, 1-13). The technical success rate of stent placement was 99%. Early adverse events were noted in 27 of 313 (8.6%) patients (perforation in 6, bleeding in 8, suprainfection in 9, other in 7). Successful endoscopic therapy was noted in 277 of 313 (89.6%) patients. When comparing the 3 groups, there was no difference in the technical success (P = .37). Early adverse events were significantly lower in the FCSEMS group compared with the DP and LAMS groups (1.6%, 7.5%, and 9.3%; P < .01). At 6-month follow-up, the rate of complete resolution of WON was lower with DP stents compared with FCSEMSs and LAMSs (81% vs 95% vs 90%; P = .001). The mean number of procedures required for WON resolution was significantly lower in the LAMS group compared with the FCSEMS and DP groups (2.2 vs 3 vs 3.6, respectively; P = .04). On multivariable analysis, DP stents remain the sole negative predictor for successful resolution of WON (odds ratio [OR], 0.18; 95% confidence interval, 0.06-0.53; P = .002) after adjusting for age, sex, and WON size. Although there was no significant difference between FCSEMSs and LAMSs for WON resolution, the LAMS was more likely to have early adverse events (OR, 6.6; P = .02).

CONCLUSIONS

EUS-guided drainage/debridement of WON using FCSEMSs and LAMSs is superior to DP stents in terms of overall treatment efficacy. The number of procedures required for WON resolution was significantly lower with LAMSs compared with FCSEMSs and DP stents.

Despite recent advances in the chemotherapy of chronic hepatitis B (CHB), an effective viral suppression after cessation of therapy has not yet been achieved. To investigate whether hepatitis B virus (HBV)-specific T-cell responses are inducible and can contribute to the viral suppression after cessation of the therapy, we conducted a proof-of-concept study with a DNA vaccine comprising of most HBV genes plus genetically engineered interleukin-12 DNA (IL-12N222L) in 12 CHB carriers being treated with lamivudine (LAM). When the ex vivo and/or cultured IFN-gamma enzyme-linked immunospot (ELISPOT) assay was performed, the detectable HBV-specific IFN-gamma secreting T-cell responses were observed at the end of treatment and during a follow-up. These type 1T-cell responses, particularly CD4(+) memory T-cell responses could be maintained for at least 40 weeks after the therapy and correlated with virological responses, but not with alanine aminotransferase elevation. Moreover, DNA vaccination under LAM treatment appeared to be well-tolerated and showed 50% of virological response rate in CHB carriers. Thus, a combination therapy of the DNA vaccine with chemotherapy may be one of new immunotherapeutic methods for the cure of CHB.

Fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) of mycobacteria play a critical role in both the physical properties and biological activities of these molecules. In a search for the acyltransferases that acylate the GPI anchors of PIM, LM, and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a putative acyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited severe growth defects and contained an increased amount of phosphatidylinositol mono- and di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides compared with the wild-type parental strain. In cell-free assays, extracts from M. smegmatis overexpressing the M. tuberculosis Rv2611c gene incorporated [14C]palmitate into acylated phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids onto 14C-labeled phosphatidylinositol mono- and di-mannosides more efficiently than extracts from the wild-type strain. Cell-free extracts from the Rv2611c mutant of M. smegmatis were greatly impaired in these respects. This work provides evidence that Rv2611c is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with the mono-mannosylated lipid acceptor being the primary substrate of the enzyme. We also provide the first evidence that two distinct pathways lead to the formation of acylated PIM2 from PIM1 in mycobacteria.

Mannose-capped lipoarabinomannans (Man-LAMs) are members of the repertoire of Mycobacterium tuberculosis modulins that the bacillus uses to subvert the host innate immune response. Interleukin-12 (IL-12) production is critical for mounting an effective immune response by the host against M. tuberculosis. We demonstrate that Man-LAM inhibits IL-12 p40 production mediated by subsequent challenge with lipopolysaccharide (LPS). Man-LAM inhibits LPS-induced IL-12 p40 expression in an IL-10-independent manner. It attenuates LPS-induced NF-kappaB-driven luciferase gene expression, suggesting that its effects are likely directly related to inhibition of NF-kappaB. This is probably because of dampening of the Toll-like receptor signaling. Man-LAM inhibits IL-1 receptor-associated kinase (IRAK)-TRAF6 interaction as well as IkappaB-alpha phosphorylation. It directly attenuates nuclear translocation and DNA binding of c-Rel and p50. Man-LAM exerts these effects by inducing the expression of Irak-M, a negative regulator of TLR signaling. Knockdown of Irak-M expression by RNA interference reinstates LPS-induced IL-12 production in Man-LAM-pretreated cells. The fact that Irak-M expression could be elicited by yeast mannan suggested that ligation of the mannose receptor by the mannooligosaccharide caps of LAM was the probable trigger for IRAK-M induction.

BACKGROUND

Lymphangioleiomyomatosis (LAM) is a rare multisystem disorder affecting primarily women of child-bearing age, and characterized by cystic lung destruction, tumors of the kidney (angiomyolipomas [AMLs]), and involvement of the axial lymphatics (lymphangioleiomyomas). Patients with LAM experience loss of pulmonary function attributed to the proliferation of abnormal-appearing smooth muscle-like cells (LAM cells). It is possible to group the LAM population by the presence or absence of extrapulmonary involvement (eg, AMLs, lymphangioleiomyomas, chylous effusions). Serum vascular endothelial growth factor (VEGF)-D, a lymphangiogenic factor, is higher in LAM patients than in healthy volunteers and has been proposed as a tool in the differential diagnosis of cystic lung disease. We assessed serum VEGF-D concentrations in relationship to clinical phenotype in LAM patients.

METHODS

Serum VEGF-D levels were quantified by enzyme immunosorbent assay for 111 patients with LAM and 40 healthy volunteers. VEGF-D levels in patients with pulmonary LAM, with or without extrapulmonary manifestations, were compared to those of healthy volunteers.

RESULTS

Serum VEGF-D levels were greater in patients with LAM compared to those of healthy volunteers (p < 0.001). However, when patient samples were grouped based on the extent of lymphatic extrapulmonary involvement (eg, lymphangioleiomyomas and adenopathy), the statistical difference was maintained only for patients with LAM with lymphatic involvement (p < 0.001), not for those patients whose disease was restricted to the lung. Serum VEGF-D levels are a good biomarker for lymphatic involvement (area under the curve [AUC], 0.845; p < 0.0001), and a fair predictor for LAM disease (AUC, 0.751; p < 0.0001). Serum levels correlated to CT scan grade (p = 0.033).

CONCLUSIONS

Serum VEGF-D concentration is a measure of lymphatic involvement in patients with LAM.

BACKGROUND

Matrix metalloproteinases (MMPs) have been shown to be involved in the pathogenesis of the destructive pulmonary lesions in patients with lymphangioleiomyomatosis (LAM); in the present report, the activation of these enzymes is examined.

OBJECTIVE

To evaluate the role of MMPs and their activating enzymes, immunohistochemical and confocalmicroscopic techniques were used to localize alpha-smooth muscle actin (alpha-SMA), HMB-45, proliferating cell nuclear antigen (PCNA), MMP-2, membrane-type 1 MMP (MT1-MMP), MT2-MMP, and MT3-MMP in lung tissues from 10 women with LAM. Tissue samples were obtained from 5 patients before treatment and in 5 patients after hormone treatment (progesterone and/or tamoxifen citrate).

RESULTS

Staining for alpha-SMA and MMP-2 was present in all the abnormal smooth muscle cells (LAM cells) in both groups. The percentages of PCNA-, MMP-2-, or MT1-MMP-positive LAM cells were much higher in the untreated group than in the treated group, whereas the percentages of HMB-45-reactive LAM cells were similar in both groups. The reactions for MT1-MMP and PCNA were preferentially localized in small spindle-shaped LAM cells; the reaction for HMB-45 was found in large epithelioid LAM cells. Many of the PCNA-positive cells were also positive for MT1-MMP. Staining for MT2-MMP and MT3-MMP was negative.

CONCLUSIONS

This study demonstrates an association between cellular proliferation and the presence of MT1-MMP in LAM cells. The activation of MMP-2 by MT1-MMP may play an important role in the destruction of lung tissue in this disorder.

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

Lymphangioleiomyomatosis (LAM) is a rare disease found primarily in white women of childbearing age. The present study describes a case of recurrent LAM after single lung transplantation. Double-staining nonisotopic in situ hybridization, immunohistochemistry, and short tandem repeat loci analysis demonstrated that the recurrent LAM lesions originated from the recipient. The data strongly support that metastatic spread of LAM cells or migration of progenitor cells plays an important role in the pathogenesis of LAM.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(alpha-L-rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(alpha-L-rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), beta-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6'-O-oleoyl-beta-D-glucopyranosyl)-beta-sitosterol (7), and beta-sitosterol-3-O-beta-D-glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein-Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.

OBJECTIVE

The Los Angeles Motor Scale (LAMS) is a brief 3-item stroke severity assessment measure designed for prehospital and Emergency Department use.

METHODS

The LAMS and NIHSS were scored in under-12-hour acute anterior circulation ischemic stroke patients. Stroke severity ratings were correlated with cervicocerebral vascular occlusion on CTA, MRA, and catheter angiography. Receiver operating curves, c statistics, and likelihood ratios were used to evaluate the predictive value for vascular occlusion of stroke severity ratings.

RESULTS

Among 119 patients, mean age was 67 (+/-18), 45% were male. Time from onset to ED arrival was mean 190 minutes (range 10 to 660). Persisting large vessel occlusions (PLVOs) were present in 62% of patients. LAMS stroke severity scores were higher in patients harboring a vascular occlusion, median 5 (IQR 4 to 5) versus 2 (IQR 1 to 3). Similarly, NIHSS stroke severity scores were higher in PLVO patients, 19 (14 to 24) versus 5 (3 to 7). ROC curves demonstrated that the LAMS was highly effective in identifying patients with PLVOs, c statistic 0.854. At the optimal threshold of 4 or higher, LAMS scores showed sensitivity 0.81, specificity 0.89, and overall accuracy 0.85. LAMS performance was comparable to NIHSS performance (c statistic 0.933). The positive likelihood ratio associated with a LAMS score>> or = 4 was 7.36 and the negative likelihood ratio 0.21.

CONCLUSIONS

Stroke severity assessed by the LAMS predicts presence of large artery anterior circulation occlusion with high sensitivity and specificity. The LAMS is a promising instrument for use by prehospital personnel to identify select stroke patients for direct transport to Comprehensive Stroke Centers capable of endovascular interventions.

Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannose-capped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.

Pulmonary lymphangioleiomyomatosis (LAM), a disease of young women, is characterized by proliferation of immature-appearing smooth-muscle cells (LAM cells) in the lungs and abdomen. LAM cells react with monoclonal antibody HMB45, which recognizes a 100-kD glycoprotein (gp100) originally found in human melanoma cells. We investigated the expression and the subcellular localization of gp100 in lung tissue from patients with LAM and in human melanoma cell lines (Malme-3M, A2058, and CHL-1), and the relationship between this expression and cellular proliferation. Binding sites for HMB45 antibody in melanoma and LAM cells were located in cytoplasmic granules resembling immature melanosomes. LAM cells reactive for proliferating-cell nuclear antigen (PCNA), a marker of cellular proliferation, were spindle-shaped, in contrast to the large, epithelioid cells reacting with HMB45 antibody. In accord with this finding, we observed an inverse relationship between the immunostaining for HMB45 antibody and PCNA in LAM and melanoma cells. Thus, LAM and melanoma cells are heterogeneous with respect to their stages of proliferation and their expression of melanoma antigens. PCNA-positive cells, which are more likely to be negative for reactivity with HMB45 antibody, may be more relevant to the progression of LAM than are HMB45-positive cells, which are the hallmark of LAM.

What is learned during perceptual learning? We address this question by analyzing how perceptual inefficiencies improve over the course of perceptual learning (Dosher & Lu, 1998). Systematic measurements of human performance as a function of both the amount of external noise added to the signal stimulus and the length of training received by the observers enable us to track changes of the characteristics of the perceptual system (e.g., internal noise[s] and efficiency of the perceptual template) as perceptual learning progresses, and, therefore, identifies the mechanism(s) underlying the observed performance improvements. Two different observer models, the linear amplifier model (LAM) and the perceptual template model (PTM), however, have led to two very different theories of learning mechanisms. Here we demonstrate the failure of an LAM-based prediction - that the magnitude of learning-induced threshold reduction in high external noise must be less or equal to that in low external noise. In Experiment 1, perceptual learning of Gabor orientation identification in fovea showed substantial performance improvements only in high external noise but not in zero or low noise. The LAM-based model was "forced" to account for the data with a combination of improved calculation efficiency and (paradoxical) compensatory increases of the equivalent internal noise. Based on the PTM framework, we conclude that perceptual learning in this task involved learning how to better exclude external noise, reflecting retuning of the perceptual template. The data provide the first empirical demonstration of an isolable mechanism of perceptual learning. This learning completely transferred to a different visual scale in a second experiment.