Retroviral vectors have induced subtle clonal skewing in many gene therapy patients and severe clonal proliferation and leukemia in some of them, emphasizing the need for comprehensive integration site analyses to assess the biosafety and genomic pharmacokinetics of vectors and clonal fate of gene-modified cells in vivo. Integration site analyses such as linear amplification-mediated PCR (LAM-PCR) require a restriction digest generating unevenly small fragments of the genome. Here we show that each restriction motif allows for identification of only a fraction of all genomic integrants, hampering the understanding and prediction of biological consequences after vector insertion. We developed a model to define genomic access to the viral integration site that provides optimal restriction motif combinations and minimizes the percentage of nonaccessible insertion loci. We introduce a new nonrestrictive LAM-PCR approach that has superior capabilities for comprehensive unbiased integration site retrieval in preclinical and clinical samples independent of restriction motifs and amplification inefficiency.

Lymphangioleiomyomatosis (LAM) is a rare, cystic lung disease that is associated with mutations in tuberous sclerosis genes, renal angiomyolipomas, lymphatic spread, and remarkable female gender restriction. The clinical course of LAM is characterized by progressive dyspnea on exertion, recurrent pneumothorax, and chylous fluid collections. Lung function declines at approximately twofold to threefold times the rate of healthy subjects, based on an annual drop in FEV1 of 75 to 120 mL in reported series. The diagnosis of pulmonary LAM can be made on high-resolution CT (HRCT) scan with reasonable certainty by expert radiologists, but generally requires a lung biopsy in cases in which tuberous sclerosis complex, angiomyolipomata, or chylous effusions are absent. The currently available treatment strategies are based on the antagonism of estrogen action, and are empiric and unproven. A trial of bronchodilators is warranted in patients with reversible airflow obstruction seen on pulmonary function testing. Pleurodesis should be performed with the initial pneumothorax, because the rate of recurrence is high. Angiomyolipomas that exceed 4 cm in size are more likely to bleed and should be evaluated for embolization. Air travel is well-tolerated by most patients with LAM. Lung transplantation is an important option for LAM patients, and can be safely performed by experienced surgeons despite prior unilateral or bilateral pleurodesis in most patients. Women with unexplained recurrent pneumothorax, tuberous sclerosis, or a diagnosis of primary spontaneous pneumothorax or emphysema in the setting of limited or absent tobacco use should undergo HRCT scan screening for LAM. Multicenter clinical trials based on several well-defined molecular targets are currently underway in the United States and Europe.

Detection of Mycobacterium tuberculosis antigens in urine is attractive as a potential means of diagnosing tuberculosis (TB) regardless of the anatomical site of disease. The most promising candidate antigen is the cell wall lipopolysaccharide antigen lipoarabinomannan (LAM), which has been used to develop commercially available enzyme-linked immunosorbent assays. Although highly variable diagnostic accuracy has been observed in different clinical populations, it is now clear that this assay has useful sensitivity for diagnosis of HIV-associated TB in patients with advanced immunodeficiency and low CD4 cell counts. Thus, this assay is particularly useful when selectively used among patients enrolling in antiretroviral treatment services or in HIV-infected patients requiring admission to hospital medical wards. These are the very patients who have the highest mortality risk and who stand to gain the most from rapid diagnosis, permitting immediate initiation of TB treatment. A recently developed low-cost, lateral-flow (urine 'dip-stick') format of the assay provides a result within 30 minutes and is potentially a major step forward as it can be used at the point-of-care, making the possibility of immediate diagnosis and treatment a reality. This paper discusses the likely utility of this point-of-care assay and how it might best be used in combination with other diagnostic assays for TB. The many further research studies that are needed on this assay are described. Consideration is particularly given to potential reasons for the variable specificity observed in existing field evaluations of LAM ELISAs. Whether this might be related to the assay itself or to the challenges associated with study design is discussed.

Lymphangiomyomatosis (LAM) is characterized by the proliferation of abnormal smooth muscle cells and cystic degeneration of the lung. LAM affects almost exclusively young women. Although lung transplantation provides effective therapy for end-stage LAM, there are reports of LAM recurrence after lung transplantation. Whether these recurrent LAM cells arise from the patient or the lung transplant donor is an area of controversy. We used microsatellite marker fingerprinting and TSC2 gene mutational analysis to study a patient with recurrent LAM after single-lung transplantation. The DNA microsatellite marker pattern indicated the presence of patient-derived LAM cells in the allograft. A somatic one base pair deletion in exon 18 of the TSC2 gene was identified in pulmonary and lymph node LAM cells before transplantation. The same mutation was in the recurrent LAM, demonstrating that the recurrent LAM was derived from the patient. Fluorescence in situ hybridization revealed that cells immunoreactive with the monoclonal antibody HMB-45 did not contain a Y chromosome. These data indicate that histologically benign LAM cells can migrate or metastasize in vivo to the transplanted lung. In addition, the patient had no evidence of a renal angiomyolipoma at autopsy and therefore demonstrated for the first time that somatic TSC2 mutations cause LAM in patients without angiomyolipomas.

Infection of cells with herpes simplex virus type 1 (HSV-1) results in a rapid alteration of phosphorylation on the large subunit of cellular RNA polymerase II (RNAP II), most likely on its C-terminal domain (S. A. Rice, M. C. Long, V. Lam, C. A. Spencer, J. Virol. 68:988-1001, 1994). This phosphorylation modification generates a novel form of the large subunit which we have designed IIi. In this study, we examine roles that HSV-1 gene products play in this process. An HSV-1 mutant defective in the immediate-early transcriptional activator protein ICP4 is able to efficiently induce IIi. Viruses having mutations in the genes for the ICP0, ICP6, or ICP27 proteins are also competent for IIi formation. In contrast, 22/n199, an HSV-1 mutant which contains a nonsense mutation in the gene encoding the immediate-early protein ICP22, is significantly deficient in IIi induction. This effect is seen in Vero cells, where 22/n199 grows relatively efficiently, and in human embryonic lung (HEL) cells, where 22/n199 growth in more restricted. RNAP II is recruited into viral replication compartments in 22/n199-infected cells, indicating that altered phosphorylation of RNAP II is not a prerequisite for nuclear relocalization of RNAP II. In addition, we show by nuclear run-on transcription analysis that viral gene transcription is deficient in HEL cells infected with 22/n199. Viral late gene transcription does not occur efficiently, and antisense transcription throughout the genome is diminished compared with that of the wild-type HSV-1 infection. These transcriptional effects cannot be explained by differences in viral DNA replication, since 22/n199 replicates its DNA efficiently in HEL cells. Our results demonstrated that ICP22 is necessary for virus-induced aberrant phosphorylation of RNAP II and for normal patterns of viral gene transcription in certain cell lines.

Lymphangiomyomatosis (LAM) is a rare disease, of unknown etiology, affecting women almost exclusively. Lung transplantation is the only consistently effective therapy for LAM. Microscopically, LAM consists of a diffuse proliferation of smooth muscle cells. LAM can occur without evidence of other disease (referred to as "sporadic LAM") or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome characterized by seizures, mental retardation, and tumors in the brain, heart, skin, and kidney. Renal angiomyolipomas occur in approximately 50% of sporadic LAM patients and in 70% of TSC patients. Loss of heterozygosity (LOH) in the chromosomal region for the TSC2 gene occurs in 60% of TSC-associated angiomyolipomas. Because of the similar pulmonary and renal manifestations of TSC and sporadic LAM, we hypothesized that LAM and TSC have a common genetic basis. We analyzed renal angiomyolipomas, from 13 women with sporadic LAM, for LOH in the regions of the TSC1 (chromosome 9q34) and TSC2 (chromosome 16p13) genes. TSC2 LOH was detected in seven (54%) of the angiomyolipomas. We also found TSC2 LOH in four lymph nodes from a woman with retroperitoneal LAM. No TSC1 LOH was found. Our findings indicate that the TSC2 gene may be involved in the pathogenesis of sporadic LAM. However, genetic transmission of LAM has not been reported. Women with LAM may have low-penetrance germ-line TSC2 mutations, or they may be mosaic, with TSC2 mutations in the lung and the kidney but not in other organs.

We have characterized the CD1b-mediated presentation pathway for the mycobacterial lipoglycan lipoarabinomannan (LAM) in monocyte-derived antigen-presenting cells. The macrophage mannose receptor (MR) was responsible for uptake of LAM. Antagonism of MR function inhibited both the internalization of LAM and the presentation of this antigen to LAM-reactive T cells. Intracellular MRs were most abundant in early endosomes, but they also were located in the compartment for MHC class II antigen loading (MIIC). Internalized LAM was transported to late endosomes, lysosomes, and MIICs. MRs colocalized with CD1b molecules, suggesting that the MR could deliver LAM to late endosomes for loading onto CD1b. LAM and CD1b colocalized in organelles that may be sites of lipoglycan antigen loading. This pathway links recognition of microbial antigens by a receptor of the innate immune system to the induction of adaptive T cell responses.

Lymphangioleiomyomatosis (LAM) is a rare progressive lung disease of women. LAM is caused by mutations in the tuberous sclerosis genes, resulting in activation of the mTOR complex 1 signaling network. Over the past 11 years, there has been remarkable progress in the understanding of LAM and rapid translation of this knowledge to an effective therapy. LAM pathogenic mechanisms mirror those of many forms of human cancer, including mutation, metabolic reprogramming, inappropriate growth and survival, metastasis via blood and lymphatic circulation, infiltration/invasion, sex steroid sensitivity, and local and remote tissue destruction. However, the smooth muscle cell that metastasizes, infiltrates, and destroys the lung in LAM arises from an unknown source and has an innocent histological appearance, with little evidence of proliferation. Thus, LAM is as an elegant, monogenic model of neoplasia, defying categorization as either benign or malignant.

Lipoarabinomannans (LAM) and lipomannans (LM) are integral parts of the mycobacterial cell wall recognized by cells involved in the innate immune response and have been found to modulate the cytokine response. Typically, mannosylated LAM from pathogenic mycobacteria have been reported to be anti-inflammatory, whereas phosphoinositol-substituted LAM from nonpathogenic species are proinflammatory molecules. In this study, we show that LM from several mycobacterial species, including Mycobacterium chelonae, Mycobacterium kansasii, and Mycobacterium bovis bacillus Calmette-Guérin, display a dual function by stimulating or inhibiting proinflammatory cytokine synthesis through different pathways in murine primary macrophages. LM, but none of the corresponding LAM, induce macrophage activation characterized by cell surface expression of CD40 and CD86 and by TNF and NO secretion. This activation is dependent on the presence of Toll-like receptor (TLR) 2 and mediated through the adaptor protein myeloid differentiation factor 88 (MyD88), but independent of either TLR4 or TLR6 recognition. Surprisingly, LM exerted also a potent inhibitory effect on TNF, IL-12p40, and NO production by LPS-activated macrophages. This TLR2-, TLR6-, and MyD88-independent inhibitory effect is also mediated by LAM from M. bovis bacillus Calmette-Guérin but not by LAM derived from M. chelonae and M. kansasii. This study provides evidence that mycobacterial LM bear structural motifs susceptible to interact with different pattern recognition receptors with pro- or anti-inflammatory effects. Thus, the ultimate response of the host may therefore depend on the prevailing LM or LAM in the mycobacterial envelope and the local host cell receptor availability.

BACKGROUND

Tenofovir (TDF) is an adenosine nucleotide analogue that has been approved for the treatment of HIV-1 infection. It also shows activity against hepatitis B virus (HBV) in patients with or without lamivudine (LAM)-associated mutations. Development of clinical or virological HBV breakthrough during TDF therapy has not been reported so far. The aim of this study was to analyse the HBV polymerase (pol) from HIV/HBV-coinfected patients with detectable serum levels of HBV DNA during treatment with TDF for longer than 6 months.

METHODS

The HBV pol was sequenced from 43 patient's serum before and during TDF therapy. Phenotypic analyses were performed using HBV replication-competent plasmids carrying unique mutations selected under TDF therapy.

RESULTS

Mean exposure to LAM was 35.3 +/- 27.5 months and to TDF 11.2 +/- 6.7 months. Genotypic analyses from 21 of the patients revealed LAM-associated mutations, and a further two patients developed a novel mutation, rtA194T, along with LAM-resistance-associated mutations. Phenotypic analyses revealed that constructs harbouring rtA194T combined with rtL180M and rtM204V displayed an over 10-fold increase in the IC50 for TDF compared with the wild type.

CONCLUSIONS

The selection of HBV mutations in HBV/HIV-coinfected patients failing TDF therapy is an unlikely event within the first 12 months of treatment. However, HBV from two of the 43 patients treated with TDF for more than 12 months was found to contain one novel mutation located distal to the catalytic site of the HBV pol. In vitro, rtA194T conferred a reduced susceptibility to TDF in the presence of LAM-associated mutations.

Over the last decade, the active role of the microenvironment in the pathogenesis of B cell lymphomas has been recognized, delivering signals that favor clonal expansion and drug resistance. We are only beginning to understand the complex cross talk between neoplastic B cells and the tissue microenvironment, for example in secondary lymphoid organs, but some key cellular and molecular players have emerged. Mesenchymal stromal cells, nurselike cells (NLC) and lymphoma-associated macrophages (LAM), in concert with T cells, natural killer cells and extracellular matrix components participate in the dialog with the neoplastic B cells. B cell receptor signaling, activation via TNF family members (i.e. BAFF, APRIL), and tissue homing chemokine receptors and adhesion molecules are important in the interaction between malignant B cells and their microenvironment. Disrupting this cross talk is an attractive novel strategy for treating patients with B cell malignancies. Here, we summarize the cellular and molecular interactions between B cell lymphoma/leukemia cells and their microenvironment, and the therapeutic targets that are emerging, focusing on small molecule inhibitors that are targeting B cell receptor-associated kinases SYK, BTK, and PI3Ks, as well as on immunomodulatory agents and T cell mediated therapies. Clinically relevant aspects of new targeted therapeutics will be discussed, along with an outlook into future therapeutic strategies.

A predominantly pig-to-human zoonotic infection caused by the novel Nipah virus emerged recently to cause severe morbidity and mortality in both animals and man. Human autopsy studies showed the pathogenesis to be related to systemic vasculitis that led to widespread thrombotic occlusion and microinfarction in most major organs especially in the central nervous system. There was also evidence of extravascular parenchymal infection, particularly near damaged vessels (Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, Guarner J, Goldsmith CS, Chua KB, Lam SK, Tan CT, Goh KJ, Chong HT, Jusoh R, Rollin PE, Ksiazek TG, Zaki SR, Nipah Virus Pathology Working Group: Nipah virus infection: Pathology and pathogenesis of an emerging paramyxoviral zoonosis. Am J Pathol 2002, 161:2153-2167). We describe here a golden hamster (Mesocricetus auratus) model that appears to reproduce the pathology and pathogenesis of acute human Nipah infection. Hamsters infected by intranasal or intraperitoneal routes died within 9 to 29 days or 5 to 9 days, respectively. Pathological lesions were most severe and extensive in the hamster brain. Vasculitis, thrombosis, and more rarely, multinucleated endothelial syncytia, were found in blood vessels of multiple organs. Viral antigen and RNA were localized in both vascular and extravascular tissues including neurons, lung, kidney, and spleen, as demonstrated by immunohistochemistry and in situ hybridization, respectively. Paramyxoviral-type nucleocapsids were identified in neurons and in vessel walls. At the terminal stage of infection, virus and/or viral RNA could be recovered from most solid organs and urine, but not from serum. The golden hamster is proposed as a suitable model for further studies including pathogenesis studies, anti-viral drug testing, and vaccine development against acute Nipah infection.

Lymphangioleiomyomatosis (LAM) is a progressive lung disease affecting almost exclusively women. The reasons for this strong gender predisposition are poorly understood. Renal angiomyolipomas occur in 50-60% of sporadic LAM patients. The smooth muscle cells of pulmonary LAM and renal angiomyolipomas are nearly indistinguishable morphologically. Here, we report the first successful cell culture of a LAM-associated renal angiomyolipoma. The cells carried inactivating mutations in both alleles of the TSC2 gene and expressed estrogen receptor , estrogen receptor , and androgen receptor. To elucidate the cellular pathways through which steroid hormones influence LAM pathogenesis, we treated the cells with both estradiol and tamoxifen. Cell growth was stimulated by estradiol, associated with phosphorylation of p44/42 MAPK at 5 min and an increase in c-myc expression at 4 h. Tamoxifen citrate also stimulated cell growth, associated with increased phosphorylation of p44/42 MAPK and expression of c-myc, indicating that tamoxifen has agonist effects on angiomyolipoma cells. This response to tamoxifen in human angiomyolipoma cells differs from prior studies of Eker rat leiomyoma cells, possibly reflecting cell type or species differences in cells lacking tuberin. Our data provide the first evidence that estradiol stimulates the growth of angiomyolipoma cells, that tamoxifen has agonist effects in angiomyolipoma cells, and that estradiol and tamoxifen impact both genomic and nongenomic signaling pathways in angiomyolipoma cells. The responsiveness of angiomyolipoma cells to estradiol may be related to the underlying reasons that LAM affects primarily women.

The granulomatous immune response in tuberculosis is characterized by delayed hypersensitivity and is mediated by various cytokines released by the stimulated mononuclear phagocytes, including tumor necrosis factor-alpha (TNF alpha) and IL-1 beta. We have demonstrated that Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM), mycobacterial heat shock protein-65 kD, and M. tuberculosis culture filtrate, devoid of LPS as assessed by the Amebocyte Lysate assay, stimulate the production of TNF alpha and IL-1 beta proteins and mRNA from mononuclear phagocytes (THP-1 cells). The effect of LAM on the release of these cytokines was specific, as only LAM stimulation was inhibited by anti-LAM monoclonal antibody. Interestingly, we found that LAM and Gram-negative bacterial cell wall-associated endotoxin LPS may share a similar mechanism in their stimulatory action as demonstrated by inhibition of TNF alpha and IL-1 beta release by monoclonal antibodies to CD14. Anti-CD14 monoclonal antibody MY4 inhibited both TNF alpha and IL-1 beta release with LAM and LPS but no effect was observed with other mycobacterial proteins. An isotype antibody control did not inhibit release of cytokines under the same experimental conditions. M. tuberculosis and its components upregulated IL-1 beta and TNF alpha mRNAs in THP-1 cells. Nuclear run-on assay for IL-1 beta demonstrated that LAM increased the transcription rate. The induction of IL-1 beta was regulated at the transcriptional level, in which these stimuli acted through cis-acting element(s) on the 5' flanking region of the IL-1 beta genomic DNA. M. tuberculosis cell wall component LAM acts similarly to LPS in activating mononuclear phagocyte cytokine TNF alpha and IL-1 beta release through CD14 and synthesis at the transcriptional level; both cytokines are key participants in the host immune response to tuberculosis.

Lymphangioleiomyomatosis (LAM) and multifocal micronodular pneumocyte hyperplasia (MMPH) produce cystic and nodular disease, respectively, in the lungs of patients with tuberous sclerosis. The objective of this study was to prospectively characterize the prevalence, clinical presentation, and genetic basis of lung disease in TSC. We performed genotyping and computerized tomographic (CT) scanning of the chest on 23 asymptomatic women with tuberous sclerosis complex (TSC). Cystic pulmonary parenchymal changes consistent with LAM were found in nine patients (39%). These patients tended to be older than cyst-negative patients (31.9 +/- 7.6 yr versus 24.8 +/- 11.6 yr, p = 0.09). There was no correlation between presence of cysts and tobacco use, age at menarche, history of pregnancy, or estrogen-containing medications. Three of the cyst-positive patients had a prior history of pneumothorax. Pulmonary function studies revealed evidence of gas trapping but normal spirometric indices in the cyst-positive group. All nine cyst-positive patients had angiomyolipomas (AML), which were larger (p < 0.05) and more frequently required intervention (p = 0.08) than cyst-negative patients (8 of 14 with AMLs, p < 0.05). Ten patients (43%) had pulmonary parenchymal nodules. Pulmonary nodules were more common in women with cysts (78% versus 21%, p < 0.05), and 52% of all patients had either cystic or nodular changes. TSC2 mutations were identified in all cyst-positive patients who were tested (n = 8), whereas both TSC1 and TSC2 mutations were found in patients with nodular disease. Correlation of the mutational and radiographic data revealed one pair of sisters who were discordant for cystic disease, two mother- daughter pairs who were discordant for nodular disease, and no clear association between cyst development and a specific mutational type. This prospective analysis demonstrates that cystic and nodular pulmonary changes consistent with LAM and MMPH are common in women with TSC.

Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes macrophages by modulating properties of the Mycobacterium-containing phagosome. Mycobacterial phagosomes do not fuse with late endosomal/lysosomal organelles but retain access to early endosomal contents by an unknown mechanism. We have previously reported that mycobacterial phosphatidylinositol analog lipoarabinomannan (LAM) blocks a trans-Golgi network-to-phagosome phosphatidylinositol 3-kinase-dependent pathway. In this work, we extend our investigations of the effects of mycobacterial phosphoinositides on host membrane trafficking. We present data demonstrating that phosphatidylinositol mannoside (PIM) specifically stimulated homotypic fusion of early endosomes in an ATP-, cytosol-, and N-ethylmaleimide sensitive factor-dependent manner. The fusion showed absolute requirement for small Rab GTPases, and the stimulatory effect of PIM increased upon partial depletion of membrane Rabs with RabGDI. We found that stimulation of early endosomal fusion by PIM was higher when phosphatidylinositol 3-kinase was inhibited by wortmannin. PIM also stimulated in vitro fusion between model phagosomes and early endosomes. Finally, PIM displayed in vivo effects in macrophages by increasing accumulation of plasma membrane-endosomal syntaxin 4 and transferrin receptor on PIM-coated latex bead phagosomes. In addition, inhibition of phagosomal acidification was detected with PIM-coated beads. The effects of PIM, along with the previously reported action of LAM, suggest that M. tuberculosis has evolved a two-prong strategy to modify its intracellular niche: its products block acquisition of late endosomal/lysosomal constituents, while facilitating fusion with early endosomal compartments.

Photoreceptor nuclei in the Drosophila eye undergo developmentally regulated migrations. Nuclear migration is known to require the perinuclear protein Klarsicht, but the function of Klarsicht has been obscure. Here, we show that Klarsicht is required for connecting the microtubule organizing center (MTOC) to the nucleus. In addition, in a genetic screen for klarsicht-interacting genes, we identified Lam Dm(0), which encodes nuclear lamin. We find that, like Klarsicht, lamin is required for photoreceptor nuclear migration and for nuclear attachment to the MTOC. Moreover, perinuclear localization of Klarsicht requires lamin. We propose that nuclear migration requires linkage of the MTOC to the nucleus through an interaction between microtubules, Klarsicht, and lamin. The Klarsicht/lamin interaction provides a framework for understanding the mechanistic basis of human laminopathies.

A number of exported proteins in E. coli, both periplasmic proteins and proteins of the outer membrane, were examined to determine when removal of the "signal sequence" occurs in vivo. One protein was processed entirely cotranslationally (amp C beta-lactamase) and one was processed entirely post-translationally (TEM beta-lactamase). The others (maltose-binding protein, arabinose-binding protein, omp A protein, lam B protein and alkaline phosphatase) showed both modes of processing, although the amount of cotranslational processing varied considerably among the individual proteins of this class. When processing occurred cotranslationally, the proteolytic removal of the "signal" was a late event. For four of the proteins studied, processing was initiated only after the polypeptides had been elongated to approximately 80% of their full length.

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3'OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3'OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3'OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNP assembly by hLARP7/PIP7S). Analyses of other LARP family members suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA-related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs.

Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-beta-estradiol (E(2)) causes a 3- to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E(2)-induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, CI-1040. In vitro, E(2) inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E(2) enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with CI-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEK-dependent pathways by E(2) leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.

OBJECTIVE

To determine the frequency of pulmonary lymphangioleiomyomatosis (LAM), a rare cystic lung disorder that occurs almost exclusively in women of reproductive age, in women with tuberous sclerosis complex (TSC), an inheritable multiorgan hamartomatosis.

METHODS

In this retrospective cohort study, the medical records of 78 women with definite TSC were reviewed, and pertinent information was recorded, including the demographic data, clinical manifestations, results of lung biopsies and autopsies, and findings on imaging studies of the chest and abdomen. All available computed tomographic (CT) scans of the chest and abdomen were reviewed.

RESULTS

Of 78 women with definite TSC seen from 1977 to 1998, 20 (26%) had evidence of LAM. Surgical lung biopsy or autopsy in 7 patients confirmed the diagnosis of their lung disease. Characteristic CT findings of LAM were noted in 13 additional patients. Twelve of these 20 patients with TSC-associated LAM had respiratory symptoms, including exertional shortness of breath and spontaneous pneumothorax, that eventually led to their pulmonary diagnosis.

CONCLUSIONS

The frequency of lung involvement (LAM) in women with TSC is substantially higher than previously suspected and may be even higher than reported in this retrospective study. These findings support the recommendation for a screening CT of the chest for all women with TSC.

Lymphangioleiomyomatosis (LAM) is a multisystem disorder of women, characterized by cystic degeneration of the lungs, renal angiomyolipomas (AML), and lymphatic abnormalities. LAM lesions result from the proliferation of benign-appearing, smooth muscle-like LAM cells, which are characterized by loss of heterozygosity (LOH) of one of the tuberous sclerosis complex (TSC) genes. LAM cells are believed to migrate among the involved organs. Because of the apparently metastatic behavior of LAM, we tried to isolate LAM cells from body fluids. A cell fraction separated by density gradient centrifugation from blood had TSC2 LOH in 33 of 60 (55%) LAM patients. Cells with TSC2 LOH were also found in urine from 11 of 14 (79%) patients with AML and in chylous fluid from 1 of 3 (33%) patients. Identification of LAM cells with TSC2 LOH in body fluids was not correlated with severity of lung disease or extrapulmonary involvement and was found in one patient after double lung transplantation. These studies are compatible with a multisite origin for LAM cells. They establish the existence of disseminated, potentially metastatic LAM cells through a relatively simple, noninvasive procedure that should be valuable for molecular and genetic studies of somatic mutations in LAM and perhaps other metastatic diseases.

Previously we had demonstrated that the termini of the arabinan component of mycobacterial cell wall arabinogalactan, the site of mycolic acid location, consists mostly of clusters of a pentaarabinofuranoside, [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2----(3 and 5)-alpha-D-Araf. Subsequently, the same arrangement was shown to dominate the non-reducing ends of lipoarabinomannan (LAM), a key component in the interaction of mycobacteria with host cell. Accordingly, we had proposed that mycobacteria universally elaborate the same Araf-containing motifs in two settings for different pathophysiological purposes. However, we now report that the termini of LAM from the virulent, Erdman, strain of Mycobacterium tuberculosis, unlike those from the attenuated H37Ra strain, are extensively capped with mannosyl (Manp) residues, either a single alpha-D-Manp, a dimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp), or a trimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp ). The use of monoclonal antibodies demonstrates a clear difference in the antigenicity of the basic and mannose-capped LAM. The possibility that these structures are a factor in the virulence of some strains of M. tuberculosis and represent an example of carbohydrate mimicry in mycobacterial infections is discussed.

Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.