HIV insertions within and proximal to host cell genes are a common finding in tissues containing high levels of HIV DNA and macrophage-associated p24 antigen expression.

HIV integration within host cell genomic DNA is a requisite step of the viral infection cycle. Yet, characteristics of the sites of provirus integration within the host genome remain obscure. The authors present evidence that in diseased tissues showing a high level of HIV DNA and macrophage-associated HIV p24 antigen expression from end stage forms of HIV disease, HIV-1 integration sites were favored within genes and transcriptionally active host cell genomic loci. Using an inverse PCR (IPCR) technique that identified dominant integrated forms of HIV, clonal IPCR products were isolated from AIDS dementia, AIDS lymphoma, and angioimmunoblastic lymphadenopathy tissues. Thirty of 34 disease-associated HIV-1 insertions were identified within annotated and hypothetical genes, an unexpected but highly nonrandom genetic coding region association (p <.026). The 1% sensitivity thresholds used for HIV IPCR suggested some form of selective expansion of cells containing these HIV proviruses. Consistent with this interpretation were the HIV-1 insertion sites identified within introns of genes that encoded for factors associated with signal transduction, apoptosis, and transcription regulation. In addition, HIV-1 proviruses were frequently found proximal to genes that encoded for receptor-associated, signal transduction-associated, transcription-associated, and translation-associated proteins. HIV-1 integration within host cell genomic DNA potentially represents a significant insertional mutagenic event. In certain cases, provirus insertions may mediate the dysregulation of specific gene expression events, providing mechanisms contributing to the pathogenesis associated with certain AIDS-related diseases.

A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages.

Primary cultured human macrophages are difficult to transfect. We have developed a DEAE-dextran DNA transfection method that mediates the reproducible transfection of primary cultured adherent human macrophages. Three factors essential for successful transfection were identified: DEAE-dextran concentration, the quantity of DNA per transfection and the incubation time of the macrophages with the transfection medium. Maximum levels of luciferase expression were attained within 24 h and maintained for at least 56 h after transfection. While serum in the transfection medium attenuated transfection, the treatment of the macrophages with chloroquine, DMSO, or glycerol did not enhance transfection within this system. A CMV enhancer/promoter mediated substantially greater luciferase expression in the macrophages than either HIV or RSV LTRs. DEAE-dextran facilitated superior transfection compared to either cationic liposome and calcium phosphate methods, and was more practical compared to electroporation for multiple transfections. This transfection protocol provides a simple, inexpensive, reproducible method for the evaluation of gene expression in primary cultured adherent human macrophages.

Polylysine enhances cationic liposome-mediated transfection of the hepatoblastoma cell line Hep G2.

Plasmid DNA condensed by polylysine enhanced cationic-liposome-mediated transfection of Hep G2 cells. The luciferase expression plasmid pCMVL was complexed with the polycation poly-L-lysine and mixed with liposomes that contained a 1:1 molar ratio of the cationic lipid 1,2-dioleoyloxy-3-trimethyl-ammoniumpropane, with the neutral phospholipid 1,2-di-(cis-9-octadecenoyl)-sn-glycero-3-phosphoethanolamine. Polylysine enhanced cationic-liposome-mediated transfection of the hepatoblastoma cell line Hep G2 9-fold compared with pCMVL complexed alone with liposomes. The ratio of cationic to anionic charge of the polylysine-pCMVL complexes, and the quantity of cationic liposomes, are important determinants for optimal transfection of Hep G2 cells.

Cationic lipid enhances in vitro receptor-mediated transfection.

The efficiency of cell-specific transfection by receptor-mediated uptake is improved by the use of cationic lipids. Asialoglycoprotein (AP) was conjugated to poly-L-lysine (PL) and complexed with the plasmid pCMVL that contains a luciferase reporter gene. The asialoglycoprotein-poly-L-lysine:pCMVL (AP-PL:pCMVL) complexes then were mixed with the cationic lipid dioctadecylamidoglycylspermine (DOGS). This complex was taken up by the hepatocyte-like cell line, Hep G2, via the asialoglycoprotein receptor. The expression of luciferase in cells transfected with the DOGS/AP-PL: pCMVL complexes were significantly increased compared with AP-PL:pCMVL complexes without DOGS. The ratio of AP-PL to DOGS is an important determinant for both transfection efficiency and for maintaining receptor specificity. Therefore, cationic lipids significantly increased the efficiency of asialoglycoprotein receptor mediated transfection in the hepatoblastoma cell line, Hep G2. The use of cationic lipids with receptor-mediated gene delivery systems could potentially increase transfection efficiency yet maintain cell-target specificity.

Atypical pulmonary and neurologic complications of amiodarone in the same patient. Report of a case and review of the literature.

Atypical manifestations of pulmonary toxicity and previously unreported autonomic nervous system dysfunction complicating amiodarone therapy were observed in a patient being treated for sustained ventricular tachycardia. Pulmonary and hepatic nodules on computed tomographic scan masquerading as metastatic carcinoma were initially noted. Focal infiltrates and a large left pleural effusion mimicking infection, malignant neoplasm, or collagen vascular disease became manifest at a later stage. Autonomic dysfunction presented as incapacitating orthostatic hypotension and persisted for six weeks after amiodarone withdrawal. The pleuropulmonary toxic effects were reversible on discontinuation of amiodarone therapy, and resolution was hastened by short-course steroid treatment.

Type specificity of complement-fixing antibody against herpes simplex virus type 2 AG-4 early antigen in patients with asymptomatic infection.

We evaluated the type specificity of complement-fixing (CF) antibody against the AG-4 early antigen of herpes simplex virus (HSV) type 2 (HSV-2) by comparing a commercial AG-4 CF kit (Simplex-2; Gene Link Australia, Inc., Princeton, N.J.) with quantal microneutralization (MN) and absorption-Western blotting in testing sera from patients with and without a history of genital herpes. Sera characterized as HSV type 1 (HSV-1) or HSV-2 positive or negative by MN were selected and tested by CF, and those with discordant results were further analyzed for specific antibodies by absorption with HSV-1 or HSV-2 antigen and Western blotting with heterologous HSV proteins. A total of 34 of 42 (81%) sera HSV-2 positive by MN, 19 of 43 (44%) sera HSV-1 positive by MN, and 0 of 19 sera negative by MN were positive by CF. Absorption-Western blotting showed that 12 of 18 (67%) sera HSV-1 positive by MN but positive by CF had no HSV-2-specific antibody and that all 7 sera HSV-2 positive by MN but negative by CF had HSV-2-specific antibody. When MN and absorption-Western blotting data were combined to analyze patients with no history of genital herpes, 7 of 19 (37%) with no HSV-2-specific antibody were positive by CF, and 7 of 27 (26%) with HSV-2-specific antibody were negative by CF. The positive and negative predictive values for the CF test were 78 and 75%, respectively, in this group. The presence of antibody to the HSV AG-4 antigen does not discriminate sufficiently between HSV-1- and HSV-2-infected patients to be of value in predicting HSV-2 infection in the absence of symptomatic disease.

Characterization of Haemophilus influenzae type b fimbriae.

We confirmed that the fimbriae of Haemophilus influenzae type b conferred hemagglutinating activity (HA) towards human erythrocytes, and erythrocytes of certain other species. Most (17/25) cerebrospinal fluid isolates lacked detectable HA on direct testing, but selective enrichment for fimbriation (f+) indicated that 22 of 25 strains could produce these surface structures. HA was unchanged from pH 4.5 to 9.5 and was not inhibited by mannose or certain other simple sugars. The HA titer of a suspension of three f+ strains was slightly decreased at 50 degrees C; HA was lost by heating at 60 degrees C for 3 min. Growth on a variety of solid and liquid media and under differing degrees of oxygenation did not change the HA titer of a suspension of three f+ strains. Fimbriation was not lost on repeated subculture. Wild-type fimbriated strains, and those derived by transformation, did not contain detectable plasmid DNA. Transformation of a strain lacking fimbriae to f+ was associated with the appearance of an outer membrane protein of 24 kilodaltons. This protein was purified from one strain to homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by selective detergent solubilization and ammonium sulfate fractionation. Colonization capacity was equivalent with an isogenic untypable strain lacking or possessing fimbriae. Fimbriae of type b H. influenzae possess characteristics similar to those structures on other gram-negative bacteria; their role in cell physiology or pathogenesis of invasive disease is unknown.

Characterization of non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae.

Ampicillin resistance in Haemophilus influenzae is most often due to the plasmid-mediated production of TEM beta-lactamase. We studied four strains with high-level ampicillin resistance (MIC of 32 micrograms/ml with an inoculum of 10(5) CFU on solid media) which did not produce detectable beta-lactamase activity with two different detection methods. Two of the four strains contained extrachromosomal DNA by agarose gel electrophoresis. Conjugation failed to transfer ampicillin resistance; in contrast, transformation yielded ampicillin-resistant transformants in three of the four strains. These transformants did not contain detectable extrachromosomal DNA. In addition, mobilization of the resistance determinant by transformation to, or conjugation with, recombination-deficient strains was unsuccessful. DNA-DNA hybridization experiments revealed no homology of the DNA of these strains with two R plasmids (one coding for ampicillin resistance, the other for chloramphenicol and tetracycline resistance). We conclude that the genetic basis of the non-beta-lactamase ampicillin resistance in these strains appears to be chromosomally mediated. We investigated the mechanism of resistance in these strains. Enzymatic modification of penicillin was not detected by autoradiography of a thin-layer chromatogram of cell sonic extracts of three ampicillin-resistant transformant strains incubated with [14C]penicillin. To assess changes in permeability of the cell envelope, a plasmid coding for beta-lactamase was conjugated into these strains, and the hydrolysis of penicillin by intact cells and cell sonic extracts was compared. Only one of three transformant strains had significantly diminished permeability. Outer membrane proteins of these strains analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed apparent differences in comparison with the isogenic ampicillin-susceptible recipient strain. Autofluorography of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Sarkosyl-solubilized crude membrane (the putative inner membranes) from these ampicillin-resistant transformant strains incubated with [3H]penicillin compared with the isogenic ampicillin-susceptible recipient strain revealed reduced binding to PBP 3 and 6, 3 and 4, or 4. In addition, affinity binding studies revealed decreased affinity of PBP 4 for ampicillin of all four transformants tested. We conclude that the major mechanism of resistance in these strains is altered penicillin-binding proteins; however, other mechanisms, including permeability, may also play a role.