A comparison between heterogeneous nuclear RNA and polysomal messenger RNA in HeLa cells by RNA-DNA hybridization.

Heterogeneous nuclear RNA (HnRNA) and mRNA from cytoplasmic polyribosomes of HeLa cells have been compared by RNA-DNA hybridization tests. 1 microg of HeLa cell DNA binds 0.05-0.10 microg of either HnRNA or mRNA. In addition, HeLa DNA that is preexposed to unlabeled HnRNA was found to have a reduced capacity to bind either HnRNA or mRNA. The results are compatible with considerable sequence similarity in the two types of RNA but, as is discussed, firm conclusions are precluded by imperfections of the hybridization reaction as presently employed.

The effect of puromycin on intranuclear steps in ribosome biosynthesis.

Inhibition of protein synthesis by puromycin (100 gamma/ml) is known to inhibit the synthesis of ribosomes. However, ribosomal precursor RNA (45S) continues to be synthesized, methylated, and processed. Cell fractionation studies revealed that, although the initial processing (45S --> 32S + 16S) occurs in the presence of puromycin, the 16S moiety is immediately degraded. No species of ribosomal RNA can be found to have emerged from the nucleolus. The RNA formed in the presence of puromycin is normal as judged by its ability to enter new ribosomal particles after puromycin is removed. This sequence of events is not a result of inhibition of protein synthesis, for cycloheximide, another inhibitor of protein synthesis, either alone or in combination with puromycin allows the completion of new ribosomes.

The turnover of nuclear DNA-like RNA in HeLa cells.

The subcellular distribution of various types of RNA in HeLa cells is described. In addition, the relative rate of synthesis of the major classes of nuclear RNA has been determined. From these experiments it can be deduced that the heterogeneous nuclear RNA fraction is rapidly synthesized and degraded within the cell nucleus.

Arrested protein synthesis in polysomes of cultured chick embryo cells.

Cells deprived of serum synthesize proteins at a reduced rate; when serum is restored the rate returns to normal. The polysomes do not dissociate, but show reduced incorporation of amino acid in vitro, and are less responsive to polyuridylic acid than are those from normal cells.

Pilot trial of infusional cyclophosphamide, doxorubicin, and etoposide plus didanosine and filgrastim in patients with human immunodeficiency virus-associated non-Hodgkin's lymphoma.

PURPOSE: To determine the following: (1) the feasibility of combining the antiretroviral didanosine (ddl) with a 96-hour continuous intravenous (IV) infusion of cyclophosphamide (800 mg/m2), doxorubicin (50 mg/m2), and etoposide (240 mg/m2) (CDE) plus filgrastim in patients with non-Hodgkin's lymphoma (NHL) associated with human immunodeficiency virus (HIV) infection; (2) the effect of ddl on CDE-induced myelosuppression and CD4 lymphopenia; and (3) the effect of CDE on serum p24 antigen and quantitative HIV blood cultures. METHODS: Twenty-five patients with HIV-related NHL received CDE every 28 or more days. Consecutive patients were assigned in an alternating fashion to group A (ddl given at a standard dose during cycles one, two, five, and six) or group B (ddl given during cycles three, four, five, and six). RESULTS: ddl use was associated with less leukopenia (mean nadir, 3.33 v 1.49 x 10(3)/microL; p = .03), neutropenia (2.38 v 1.07 x 10(3)/microL; p = .03), and thrombocytopenia (76 v 48 x 10(3)/microL; p = .059), and fewer RBC (1.6 v 3.1 per cycle; p < .01) and platelet transfusions (0.7 v 1.5 per cycle; p < .01), but had no significant effect on CD4 lymphopenia. Furthermore, lymphomatous bone marrow involvement and low CD4 count were associated with significantly greater myelosuppression. Although there was no substantial change in serum p24 antigen, the HIV blood culture became quantitatively more positive or converted from negative to positive in seven patients (64%). Complete response (CR) occurred in 58% of patients (95% confidence interval, 38% to 78%), median CR duration exceeded 18 months, tumor-related mortality was 20%, and median survival was 18.4 months. CONCLUSION: Our results suggest that the CDE and filgrastim regimen is tolerable and effective for patients with HIV-associated NHL, and that combination with ddl is feasible and may result in less myelosuppression.

Detection of HIV in fetal central nervous system tissue.

Neurological disease is a common finding in children with AIDS and in others without signs of disease but with evidence of congenital HIV-1 infection. To investigate the possibility that HIV-1 can infect fetal central nervous system (CNS) tissue and therefore possibly serve as the substrate for the abnormal neurodevelopment characteristic of pediatric AIDS, eight abortus CNS samples (one set of twins) from seven HIV-1-seropositive intravenous drug users (IVDUs) and eight control abortus CNS samples from eight HIV-1-seronegative IVDUs were analyzed for HIV-1 infection. HIV-1 nucleic acid was detected only after the use of polymerase chain reaction (PCR) in three of eight CNS samples from HIV-seropositive IVDUs but not in samples from seronegative subjects. In situ hybridization confirmed that HIV-1 DNA sequences were in cells in the CNS parenchyma of two of the three positive samples. This study demonstrates that HIV-1 can infect human fetal CNS tissue in vivo, but that the use of PCR may be necessary for its detection.

Proliferative membranopathy and human immunodeficiency virus in AIDS hearts.

In order to determine if cardiac tissue from AIDS patients or patients with seropositivity to HIV-1 might be infected by HIV-1, portions of myocardium obtained postmortem were evaluated for HIV-1 DNA sequences. Cellular DNA was extracted and digested with EcoR1 and Southern blots were performed. One of three AIDS hearts was positive for HIV-1 DNA sequences without amplification, whereas two additional hearts were positive for HIV-1 DNA after amplification. Accordingly, other tissue from the heart positive for HIV-1 without amplification was studied by electron microscopy to localize HIV virions. Unexpectedly, large numbers of proliferating multilamellated membrane bodies were identified in myocytes, predominantly associated with mitochondria. Identical membrane bodies were found in two additional AIDS hearts, and in one heart from a patient with seropositivity to the AIDS virus, but in none of three similarly fixed controls. Immunocytochemistry for HIV core (p24) and envelope (gp120) antigens did not localize gold-labeled antibodies to the membrane bodies. We believe this membranopathy may be an HIV-1- or AIDS-specific abnormality of unknown etiology that may be related to the ultimate development of cardiomyopathy. In addition, our studies provide further support that HIV-1 may be present in AIDS hearts, although as yet we cannot state with certainty where the HIV-1 is located in these tissues.

HIV-1 infection of human fetal thymocytes.

Some neonates with congenital human immunodeficiency virus type 1 (HIV-1) infection exhibit immune dysregulation. This suggests that fetal CD4+ cells, possibly thymocytes, may be infected during gestation. If thymocytes are infected, this may result in a disruption of T-cell differentiation. To examine this hypothesis, normal human fetal thymocytes were established in tissue culture, characterized, and then exposed to HIV-1. On initial isolation, fetal thymocytes were analyzed for phenotypic markers by flow cytometry and assessed for T-cell function by mitogen-stimulated thymidine incorporation. The thymocytes comprised greater than 70% double positive (CD4+, CD8+) cells and responded to T- but not to B-cell mitogens. Thereafter, thymocytes were incubated in either tissue culture medium containing infectious HIV-1 or in control (HIV-free) medium. Infection of fetal thymocytes was determined by light and electron microscopy in combination with immunocytochemistry, molecular hybridization, and an infectious cell center (ICC) assay. After 1 week in culture, the thymocytes exposed to HIV-1 were positive by immunocytochemistry for the HIV-1-associated protein gp41. In addition, the presence of HIV-1 DNA was detected by molecular hybridization confirming infection of these cells. The ICC assay demonstrated the production of infectious HIV-1 particles and budding of mature virions was observed by electron microscopy. These studies demonstrate that human fetal thymocytes can be infected with HIV-1 in vitro and that this infection results in production of infectious virions. These results support the hypothesis that vertical transmission of HIV-1 in vivo may result in the infection of fetal thymocytes, which may contribute to postnatal HIV-1-associated pathologic conditions.

Influence of vegetation size on clinical outcome of right-sided infective endocarditis.

Endocarditis involving right-sided valvular structures is largely a disease of intravenous drug abusers. The majority of these patients respond to antibiotic therapy with clearing of their bacteremia and preservation of their hemodynamic status. This study evaluated the prognostic value of echocardiographically determined vegetation size in 23 episodes of right-sided valvular endocarditis in 21 patients. Right-sided vegetations were visualized in 19 of 23 episodes (83 percent). Of these, a vegetation of 1.0 cm or greater was found in 11. No patient with an echocardiographically determined vegetation size of less than 1.0 cm required surgery, whereas four of 11 (36 percent) of those episodes in which the vegetation size was 1.0 cm or greater required surgery for persistent pyrexia (p less than 0.05). In all patients requiring surgery, a bioprosthetic tricuspid valve was placed at the time of initial surgery and in no patient did early reinfection occur. This study reconfirms the benign prognosis of right-sided valvular endocarditis. Further, although vegetations of less than 1.0 cm identify those patients who will respond to medical therapy, echocardiographically documented vegetations of 1.0 cm or greater are associated with a significantly lower response rate to appropriate medical therapy. The association of fever that persists for more than three weeks in the absence of another source of infection with an echocardiographically demonstrable right-sided vegetation of 1 cm or more identifies those patients who will require surgical intervention. Finally, tricuspid valve replacement can be performed at the time of initial surgery without undue concern for early reinfection or valve dysfunction.

Rhodotorula rubra peritonitis in patients undergoing continuous ambulatory peritoneal dialysis.

Although still uncommon, fungal peritonitis is being reported with increasing frequency in patients with renal failure who are undergoing long-term peritoneal dialysis. Although most cases have involved Candida albicans, other opportunistic pathogens have been reported as well. Recently, three patients were seen with serious peritoneal infections due to Rhodotorula rubra, a "nonpathogenic" fungus. In two patients, peritoneal fibrosis made further peritoneal dialysis impossible. The third patient died from complications of peritonitis. Environmental cultures revealed a possible common-source outbreak. Literature review reveals that infection due to this organism is most unusual and occurs only in the hospital setting in patients with serious compromise of host defenses.

Characterization of IgG and IgG subclass antibodies present in paired maternal and fetal serum which are directed against HIV-1 proteins.

Passive immunity is conferred to the fetus by maternal antibodies, the majority of which are transported across the placenta during the third trimester of pregnancy. To determine the placental transport of anti-HIV-1 antibodies, serum from 5 women infected with human immunodeficiency virus (HIV) and their abortuses were examined for anti-HIV-1 antibodies. The gestational age of the abortuses ranged from 18 to 24 weeks and following polymerase chain reaction amplification, HIV-1 gag DNA was detected in tissue from 2 of the abortuses. The concentration of total IgG antibodies present in cord blood ranged from 2.9% to 12.5% of maternal levels. Antibodies directed against the envelope proteins, gp160 and gp120, the reverse transcriptase protein, p66, and the capsular protein, p24, were present in fetal and maternal serum. Although IgG1 was the predominant subclass antibody generated in response to HIV-1 proteins, IgG2, IgG3, and IgG4 directed against HIV-1 proteins were also detected. There were large differences in the antigens recognized by the antibodies produced in the mothers, and the IgG subclasses of the antibodies produced. HIV-1 proteins recognized by antibodies present in cord blood were similar to those recognized by paired maternal serum and IgG1, IgG2, IgG3 recognizing HIV-1 proteins were detected in fetal serum. However, there was a dichotomy in placental transport of IgG subclass antibodies to HIV-1 proteins. The role of these antibodies in prevention of vertical transmission of HIV-1 has yet to be determined.

Human immunodeficiency virus-1 infection of the nervous system: an autopsy study of 268 adult, pediatric, and fetal brains.

The central nervous system (CNS) of 221 adults and 31 infants or children with the acquired immunodeficiency syndrome (AIDS) was examined with immunocytochemistry for infectious agents and for human immunodeficiency virus-1 (HIV-1) antigen (gp41). Since the major risk factor in this population was intravenous drug abuse, there were more female and pediatric patients than in other neuropathology autopsy series. Although children had a different spectrum of pathologic changes, including less frequent opportunistic infections, women did not differ from men in terms of types or incidence of opportunistic infections, vascular disease, neoplasia, and subacute AIDS encephalitis (SAE). Subacute AIDS encephalitis was detected in 26% of adult and 48% of pediatric brains. Immunocytochemical analysis of 100 adult and 20 pediatric brains revealed gp41 immunoreactivity in 78% and 40%, respectively. Virtually all adult brains with SAE had gp41 immunoreactivity in macrophages and microglia. Even brains with no significant pathology had frequent gp41 immunoreactivity, especially in the basal ganglia. In pediatric brains, including cases with SAE, gp41 immunoreactivity was less abundant, suggesting the possibility of latent infection or viral clearance. Spinal cords with vacuolar myelopathy or corticospinal tract degeneration had only rare gp41-positive cells. Brains from 16 aborted fetuses from HIV-1-seropositive women were all negative for gp41 immunoreactivity, but 12 brains were positive for HIV-1 by the polymerase chain reaction. These results may indicate that HIV-1 infection in fetal brains is below the limits of detection of immunocytochemistry. The differences noted between adults and children suggest that adults more often have productive CNS HIV-1 infection.

Immediate tricuspid valve replacement for endocarditis. Indications and results.

Tricuspid valve excision for tricuspid endocarditis in addicts is recommended to avoid early reinfection, continued sepsis, and late reinfection because of the resumption of intravenous drug abuse. Valvectomy is allegedly well tolerated hemodynamically by some, but it leads to heart failure in at least a third of patients. In our experience in 10 addicts with staphylococcal endocarditis who had failed to respond to antibiotic therapy, tricuspid valve replacement allowed all 10 to leave the hospital free of infection and free of heart failure. Resumption of drug addiction in three led to septic death, but not necessarily to tricuspid reinfection. Two returned to jobs requiring a high level of physical labor and tolerated this without difficulty. We find no need to follow the practice of tricuspid valve excision for tricuspid endocarditis in addicts. Those who refrain from drug abuse are well served by valve replacement. Those who do not are doomed with or without a tricuspid valve.

Mycobacterium marinum infection in a patient with the acquired immunodeficiency syndrome.

A variety of mycobacterial organisms may infect patients with acquired immunodeficiency syndrome. A patient with acquired immunodeficiency syndrome, lymphoma, and sporotrichoid Mycobacterium marinum is described. The patient responded completely to antimycobacterial therapy but relapsed when he discontinued his medications six months into his course. Disease persistence in spite of therapy had been noted in other immunocompromised states but not previously in acquired immunodeficiency syndrome. Patients with acquired immunodeficiency syndrome may require prolonged treatment or suppressive therapy for Mycobacterium marinum infections.

Photodegradation kinetics and transformation products of ketoprofen, diclofenac and atenolol in pure water and treated wastewater.

Pharmaceutical compounds such as ketoprofen, diclofenac and atenolol are frequently detected at relatively high concentrations in secondary effluents from wastewater treatment plants. Therefore, it is important to assess their transformation kinetics and intermediates in subsequent disinfection processes, such as direct ultraviolet (UV) irradiation. The photodegradation kinetics of these compounds using a medium pressure (MP) lamp was assessed in pure water, as well as in filtered and unfiltered treated wastewater. Ketoprofen had the highest time- and fluence-based rate constants in all experiments, whereas atenolol had the lowest values, which is consistent with the corresponding decadic molar absorption coefficient and quantum yield. The fluence-based rate constants of all compounds were evaluated in filtered and unfiltered wastewater matrices as well as in pure water. Furthermore, transformation products of ketoprofen, diclofenac and atenolol were identified and monitored throughout the irradiation experiments, and photodegradation pathways were proposed for each compound. This enabled the identification of persistent transformation products, which are potentially discharged from WWTP disinfection works employing UV photolysis.