Predictors of immunity after hepatitis A vaccination in HIV-infected persons.

Hepatitis A virus (HAV) infection remains a health risk for human immunodeficiency virus (HIV)-infected persons. While the inactivated HAV vaccine affords protection to immunocompetent persons >95% of the time, rates of developing protective antibody (anti-HAV) in HIV+ persons are considerably lower. Although low CD4+ T-cell counts have previously been reported to be correlated with this poor response, the effect of HIV viraemia on HAV vaccine response has not previously been reported. The medical records of HIV-infected patients who had received at least one dose of HAV vaccine (Havrix, 1440 EIU) were reviewed for factors associated with the development of a protective anti-HAV response. Serological data with regard to anti-HAV status after vaccination were available in 238 patients with 133 individuals (49.6%) developing immunity after vaccination. In a logistic regression model, the only factors associated with a protective antibody response were an HIV plasma RNA level <1000 copies/mL at the time of vaccination (P = 0.011) and male gender (P = 0.016). Neither nadir CD4+ T cell count nor CD4+ T-cell count at time of vaccination were predictive of the development of anti-HAV. Suppression of HIV replication at time of vaccination is associated with a protective antibody response to HAV vaccination in HIV-infected adults. The low rate of response warrants further research in alternative strategies for HAV vaccination among HIV-infected persons.

Persistent low-level viraemia and virological failure in HIV-1-infected patients treated with highly active antiretroviral therapy.

OBJECTIVE: To assess the prognostic significance of persistent low-level viraemia (PLV, defined as persistent plasma viral loads of 51-1000 HIV-1 RNA copies/mL for at least 3 months) in patients who had achieved viral suppression on antiretroviral therapy (ART). METHODS: A retrospective cohort of HIV-infected patients who received ART, were followed-up for > or =12 months, made regular visits to the clinic during which blood tests were performed for an ultrasensitive HIV RNA assay every 3 months, and achieved viral loads <50 copies/mL were evaluated. Virological failure was defined as two consecutive viral load measurements >1000 copies/mL. RESULTS: Of 362 patients, 78 (27.5%) experienced PLV. The demographics of patients with and without PLV were similar. PLV occurred at a mean (+/-standard deviation) of 22.6+/-16.9 months after ART initiation and lasted for 6.4+/-3.4 months. During a median follow-up of 29.5 months, patients with PLV had a higher rate of virological failure (39.7% vs 9.2%; P < 0.001). The median time to failure was 68.4 months [95% confidence interval (CI) 37.0-99.7] for patients with PLV and >72 months for patients without PLV (log rank test, P < 0.001). By Cox regression, patients with PLV had a greater risk of virological failure [hazard ratio (HR) 3.8; 95% CI 2.2-6.4; P < 0.001]. Among patients with PLV, a PLV of >400 copies/mL (HR 3.3; 95% CI 1.5-7.1; P = 0.003) and a history of ART (HR 2.4; 95% CI 1.0-5.7; P = 0.042) predicted virological failure. CONCLUSIONS: PLV is associated with virological failure. Patients with a PLV >400 copies/mL and a history of ART experience are more likely to experience virological failure. Patients with PLV should be considered for treatment optimization and interventional studies.

[First diagnosis of Fallot tetralogy in a 74-year-old man]. / Erstdiagnose einer Fallot-Tetralogie bei einem 74-jährigen Mann Kasuistik über einen komplexen Fall einer Fallot-Tetralogie bei einem 74-jährigen Erwachsenen, der ohne chirurgische oder medizinische Behandlung überlebte.

Tetralogy of Fallot is the most common cyanotic cardiac malformation in late childhood and adult, occurring in approximately 0.25 of 1000 live births. Most patients undergo early surgical correction. Therefore, the natural history of this disease has been evaluated in only a few cases. We report a complex case of a tetralogy of Fallot, who reached the age of 74 years without surgical or medical treatment and who was transferred to our clinic after syncope due to ventricular tachycardia.

Inhibition of adherence of Mycobacterium avium complex and Mycobacterium tuberculosis to fibronectin on the respiratory mucosa.

Mycobacterium species adhere to the respiratory mucosa via mucus and fibronectin of extracellular matrix exposed by damaged epithelium. We have investigated whether inhibiting adherence to fibronectin influences subsequent infection of human respiratory tissue by Mycobacterium avium complex and Mycobacterium tuberculosis. Human respiratory tissue was pretreated with mycobacterial fibronectin attachment proteins prior to infection with M. avium complex and M. tuberculosis and the number of recoverable bacteria over time was compared to untreated controls. Inhibition significantly reduced recovery of M. avium complex at 15min (P= 0.02), 7days (P = 0.04), and 14 days (P= 0.03); whereas recovery of M. tuberculosis was only reduced at 15 min (P = 0.01) and not at later timepoints. We conclude that M. avium complex and M. tuberculosis infection of the mucosa proceeds by different mechanisms, since M. tuberculosis infection is independent of fibronectin adherence.

Interaction of Mycobacterium tuberculosis with human respiratory mucosa.

OBJECTIVE: Endobronchial infection is associated with pulmonary tuberculosis in the majority of cases. We have investigated the adherence of Mycobacterium tuberculosis to the human respiratory mucosa. DESIGN: Organ cultures constructed with human tissue were infected with M. tuberculosis in the presence or absence of mycobacterial fibronectin attachment cell surface proteins and examined by scanning electron microscopy. RESULTS: M. tuberculosis adhered mainly to extracellular matrix (ECM) in areas of mucosal damage, but not to ciliated mucosa, intact extruded cells, basement membrane or collagen fibres. Bacteria also adhered to fibrous but not globular mucus and occasionally to healthy unciliated mucosa, open tight junctions and to extruded cells that had degenerated, exposing their contents. There was a significant reduction (p<0.05) in the number of bacteria adhering to ECM after pre-incubation of bacteria with fibronectin and after pre-incubation of the tissue with M. avium fibronectin attachment protein (FAP) and M. bovis antigen 85B protein, in a concentration dependent manner. The combined effect of FAP and antigen 85B protein was significantly greater than either protein alone. Bacterial adherence to fibrous mucus was not influenced by fibronectin. CONCLUSION: We conclude that M. tuberculosis adheres to ECM in areas of mucosal damage at least in part via FAP and antigen 85B protein.

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa.

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

Characterization of the fibronectin binding motif for a unique mycobacterial fibronectin attachment protein, FAP.

Studies were performed to define the fibronectin binding motif of the previously identified Mycobacterium avium fibronectin attachment protein (FAP-A). Using synthetic peptides of a previously identified fibronectin binding region (amino acids 269-292), the minimal binding sequence was determined to be 12 amino acids, 269-280 (FAP-A-(269-280)). Synthetic peptides were prepared in which each amino acid in the 269-280 sequence was substituted with Ala. Assessment of the effect of Ala substitution on fibronectin binding showed that the presence of Ala at amino acids 273-276 (RWFV) completely abrogated fibronectin binding activity. Furthermore, the ability to inhibit the attachment of viable Mycobacterium bovis BCG to fibronectin was abrogated by Ala substitution at the RWFV sites. To validate the function of RWFV, further studies were performed with recombinant FAP-A in which single Ala mutations were generated for the RWFV sites and as controls at amino acids 269 and 280. Mutant FAP-A containing single Ala substitutions at the RWFV sites (amino acids 273, 274, 275, or 276) showed significant abrogation of fibronectin binding function. Recombinant FAP-A with Ala substitutions at either 269 or 280 showed wild type activity. When the four essential amino acids (RWFV) were either substituted en bloc with Ala or were all deleted, complete loss of fibronectin binding function was observed. Control recombinant proteins with en bloc Ala substitutions or deletions at four positions outside the fibronectin binding region (amino acids 255-257) retained functional activity. These data show that the RWFV sequence is necessary for fibronectin binding function of FAP-A. Furthermore, the data suggest that mycobacterial FAP proteins, all of which share the RWFV binding motif, constitute a family of highly homologous proteins that bind fibronectin in a unique manner.

Antisense modulation of the ICAM-1 phenotype of a model human bone marrow stromal cell line.

Efficient stable gene transfer was achieved in a model human bone marrow stromal cell line, KM-102, using both Epstein-Barr virus and BK virus episomal expression vectors. Using this episomal expression system, effective overexpression and inhibition of ICAM-1 expression was achieved in stably transfected KM-102 cells by sense and antisense RNA gene transfer, respectively. Loss of surface ICAM-1 on antisense KM-102 transfectants did not significantly affect adhesion to LFA-1-bearing JY hematopoietic cells. However, KM-102 ICAM-1 overexpressors demonstrated enhanced binding (2.5-fold) to phorbol ester-treated, but not untreated, LFA-1-bearing JY cells. The increased binding could be blocked with anti-ICAM-1 antibodies. These findings suggest that while ICAM-1 is not required for basal adhesion between stromal and hematopoietic cells, stromal ICAM-1 may contribute to stromal:leukemic cellular interaction when bound to the phorbol ester-dependent high-avidity state of hematopoietic LFA-1.

A glycosylphosphatidylinositol-anchored cytokine can function as an artificial cellular adhesin.

A novel strategy for altering the adhesive properties of cells has been developed which is based upon the use of artificial adhesins. Specifically, a glycosylphosphatidylinositol (GPI)-modified variant of the cytokine macrophage colony stimulating factor (M-CSF), designated M-CSF.GPI, was expressed on the surface of human bone marrow stromal cells. A chimeric M-CSF:decay-accelerating factor expression construct was used for M-CSF.GPI expression. Cell:cell binding assays established that this artificially membrane-tethered cytokine functions as a potent cellular adhesin, allowing for enhanced binding to M-CSF receptor-expressing cellular transfectants. Antibody blocking analyses confirmed the M-CSF:M-CSF receptor dependence of the enhanced intercellular binding. This capacity to direct the cellular interactive repertoire of selected cells can in principle be applied to other cell types and other molecular pairs to be used in cell-based therapies.

C-FMS dependent HL-60 cell differentiation and regulation of RB gene expression.

The dependence of induced myelomonocytic cell differentiation, and regulation of the RB tumor suppressor gene during this process, on the c-fms gene product, the CSF-1 lymphokine receptor, was determined in HL-60 promyelocytic leukemia cells. Adding a monoclonal antibody with specificity for the c-fms gene product to cells treated with various inducers of myelomonocytic or macrophage differentiation, including retinoic acid and 1,25-dihydroxy vitamin D3, inhibited the rate of differentiation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greater inhibition of differentiation. In a stable HL-60 transfectant overexpressing the CSF-1 receptor at the cell surface due to a constitutively driven c-fms trans gene, the rate of differentiation was enhanced compared to the wild type cell, consistent with a positive regulatory role for the CSF-1 receptor. The anti-fms antibody caused much less inhibition of differentiation in the transfectants than in wild type cells, consistent with a larger number of receptors causing reduced sensitivity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB expression per cell, which offset the decrease normally caused by differentiation inducers (1,25-dihydroxy vitamin D3 and retinoic acid). The changes in RB expression preceded changes in the RB protein to the hypophosphorylated state. Most of the RB protein in proliferating cells was phosphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of G0 arrest. Thus the metabolic cascade leading to myelomonocytic differentiation of HL-60 cells appears to be driven by a function of the c-fms protein. Inhibiting that process by attacking this receptor impedes differentiation and also compromises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide additional support for a potential role for down regulating RB expression in promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF-1 actions.

Cloning and sequence analysis of the human parathyroid hormone gene region.

A region of 50 kb around the human PTH gene was cloned and mapped by restriction analysis. Sequence analysis was performed and 3270bp determined, completing the sequence of the gene. The nucleotide sequence was analysed with regard to homology between human, bovine and rat PTH genes, and various potential cis-acting regulatory elements were identified. The gene region lacks an obvious CpG island. The PTH gene region in patients suffering from (pseudo)-hypoparathyroidism was investigated by Southern blotting. No detectable alteration in the fragment patterns was observed. Results of segregation analysis in families with affected individuals was inconclusive.

Directional antisense and sense cDNA cloning using Epstein-Barr virus episomal expression vectors.

A set of Epstein-Barr virus (EBV) episomal expression vectors, incorporating either the Rous sarcoma virus 3' long terminal repeat or the human metallothionein IIA gene promoter, were constructed. The transcriptional cassettes encompassed by these vectors were designed to permit both antisense and sense RNA transcription. A novel methodology was developed for directional cDNA cloning using an oligodeoxyribonucleotide adapter; the EBV episomal vectors alternatively enabled the insertion of cDNA segments in antisense or sense orientations. We propose a strategy for random antisense RNA mutagenesis exploiting this vector system and a method for episome-based directional antisense cDNA cloning and expression, permitting the rapid identification of genes mediating selectable cellular functions.

Use of an Epstein-Barr virus episomal replicon for anti-sense RNA-mediated gene inhibition in a human cytotoxic T-cell clone.

A methodology was developed for stable gene transfer into cloned nontransformed human T lymphocytes. Stable high-level gene expression was achieved in cloned human T cells by using a self-replicating Epstein-Barr virus (EBV) episomal replicon. A comparison of five eukaryotic promoters established that the Rous sarcoma virus 3' long terminal repeat (RSV 3' LTR) and the lymphopapilloma virus (LPV) 5' LTR are optimal for episome-based expression in T cells. Effective (greater than 95%), selective, and reversible anti-sense RNA-mediated gene inhibition of a model T-cell-associated molecule (CD8) was achieved in a cytotoxic human T-cell clone by using an EBV episome-based, RSV 3' LTR-driven expression system. The linking of anti-sense RNA mutagenesis and T-cell cloning technologies should contribute significantly to studies of human T-cell function.

Glycolipid reanchoring of T-lymphocyte surface antigen CD8 using the 3' end sequence of decay-accelerating factor's mRNA.

Decay-accelerating factor (DAF) is one of a family of cell-associated proteins that undergo posttranslational modifications in which glycolipid anchoring structures are substituted for membrane-spanning sequences. The signals that direct the covalent substitution reaction in these proteins are unknown. Human DAF was expressed in Chinese hamster ovary (CHO) cells and murine BW lymphocytes. In both cases, the xenogeneic DAF in transfectants incorporated a glycolipid anchor. A chimeric CD8-DAF cDNA, encompassing the extra-cellular region of the T-lymphocyte surface antigen CD8 and the 3' end of DAF mRNA (encoding the C-terminal region of mature DAF as well as the hydrophobic extension peptide), was expressed in human leukemia lines after transfection with an Epstein-Barr virus-based episomal vector. The chimeric protein in transfectants demonstrated glycolipid anchoring, whereas unaltered CD8 in control experiments did not. The signals directing glycolipid anchoring in eukaryotic cells are thus evolutionarily conserved and contained in the 3' end of the DAF sequence.