Human coronavirus polyadenylated RNA sequences in cerebrospinal fluid from multiple sclerosis patients.

Human coronaviruses, represented by the two prototype strains HCV-OC43 and HCV-229E, are important human respiratory pathogens, also associated with necrotizing enterocolitis. Two previous studies, one describing the electron microscopic observation of doughnut-shaped particles, resembling coronaviruses, in a perivascular inflammatory lesion of brain tissue taken at autopsy from a multiple sclerosis patient, and the other one reporting the isolation of coronaviruses from the brains of two multiple sclerosis patients, suggested the possible association between coronaviruses and human demyelinating diseases. We analysed polyadenylated RNAs extracted from cerebrospinal fluid of twenty randomly selected multiple sclerosis patients and ten patients with other neurological diseases (medullary atrophy, Parkinson's disease, polyneuropathy, senile dementia, headache and toxic polyneuropathy) by reverse transcription and polymerase chain reaction searching for HCV-OC43 and HCV-229E sequences. By hybridization analysis of amplification products, we detected HCV-OC43 polyadenylated RNAs in ten specimens of patients with multiple sclerosis. Furthermore, we found positive hybridization signals for HCV-OC43 in the other neurological diseases, except for the toxic polyneuropathy specimen. Positivity for HCV-229E was observed in seven specimens of multiple sclerosis cerebrospinal fluid; one headache cerebrospinal fluid and the medullary atrophy specimen also resulted positive for HCV-229E. Moreover, using a solid phase technique, we report for the first time the sequence of a cDNA fragment derived from RNA extracted from cerebrospinal fluid of multiple sclerosis patient, belonging to the open reading frame which codes for the HCV-OC43 nucleoprotein N. Furthermore, cDNA sequences revealed the presence of a mixed viral population.

Pseudomonas aeruginosa proteases: purification procedures for an enzymatic standard.

Pseudomonas aeruginosa may cause severe infections in debilitated patients. Strains of this microorganism produce several extracellular space proteins, some of which are believed to be virulence factors. There are experimental correlations between the ability to produce proteases and virulence. Treatment of bacteria with subinhibitory concentrations of antimicrobial agents frequently increases bacterial phagocytosis, intracellular killing, and suppresses the production of bacterial virulence factors, including extracellular enzymes. We suggest a simple method for production, purification and quantitation of Pseudomonas aeruginosa extracellular proteases suitable for use in investigations of their role as virulence factors.

DNA probe for the human coronavirus OC43 also detects neonatal calf diarrhea coronavirus (NCDCV).

Human coronaviruses (HCV) OC43 and 229E are the second most frequently isolated agents of common colds, and have also been associated with severe upper respiratory infections in children and with gastroenteritis of unknown etiology, such as infantile necrotizing enterocolitis. While HCV-OC43 and neonatal calf diarrhea coronavirus NCDCV cannot be held responsible for enteric infection in man, serological data suggest the possible existence of a human coronavirus, antigenically related to HCV-OC43 and NCDCV, and responsible for enteric infections. We developed a rapid and sensitive method for the diagnosis of the human respiratory coronavirus infections, and for detecting these viruses in suspect coronavirus infections. This assay entails a reverse transcriptase polymerase chain reaction, followed by Southern blot analysis with a probe specific for the amplification products.

HIV type 1 intraperitoneal infection of rabbits permits early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA from peripheral blood mononuclear cells.

The aim of this study is the development of an animal model useful for studying HIV-1 pathogenesis, candidate vaccines, and antiviral drugs. Aseptic thioglycolate peritonitis was induced in six rabbits. After 4 days, four rabbits were infected with 1 ml of HIV-1 stock containing 100 times the MID50. Blood samples were collected every 2 weeks for 8 months. Serum antibodies were tested by ELISA, using as antigen the recombinant protein p24; synthetic peptides of highly conserved regions of p31, gp41, and gp120; and a synthetic peptide of gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Furthermore, neutralizing antibodies were tested by a microscale neutralization assay. Proviral DNA was detected by PCR, and virus isolation was performed by a cocultivation technique using primary rabbit peripheral blood mononuclear cells (PBMCs). All infected rabbits produced antibodies to HIV-1 proteins within 2 weeks and up to 8 months after virus infection. Serum antibodies were directed against the Env (gp120 and gp41), Gag (p24), and Pol (p31) proteins and against two synthetic peptides whose sequence corresponds to gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Neutralizing antibodies were also detected in the sera of infected animals. Proviral DNA was detected in PBMCs by PCR within 4 weeks and up to 8 months after HIV-1 infection. HIV-1 was also isolated from PBMCs of infected animals at 30, 60, and 120 days after infection. Results obtained indicate that HIV-1 intraperitoneal infection of the rabbit permits the early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA sequences from PBMCs.

Systemic hyperthermia in the treatment of HIV-related disseminated Kaposi's sarcoma. Long-term follow-up of patients treated with low-flow extracorporeal perfusion hyperthermia.

Treatment of HIV and related malignancies with pharmacologic and biologic agents has not appreciably modified the course of disease. Immunologic impairment remains the critical factor in response. We report the long-term results of a single session of low-flow (0.3 L/min) extracorporeal perfusion hyperthermia on 29 men and 2 women with disseminated Kaposi's sarcoma and profound immunologic impairment. Any antiretroviral drug employed by the patient was stopped 72 hours prior to treatment and withheld during the period of follow-up. Core temperature was raised to 42 degrees C and held for 1 hour with extracorporeal perfusion and ex vivo blood heating to 49 degrees C as the means of temperature control. Of 31 patients, 2 died of complications secondary to treatment (cardiac arrhythmia; CNS bleed). There were two cases of intravascular coagulopathy. Pressure point skin damage may occur despite adequate cushioning. At 30 days posttreatment complete or partial regressions were seen in 20/29 of those treated, with regressions persisting in 14/29 of those treated by 120 days posttreatment. At 360 days, 4/29 maintain tumor regressions with 1 in complete remission (at 26 months). The patient in complete remission remains culture-negative and PCR-negative for HIV. CD4+ counts rose from around 250 to, and remain around, 800 in this man. Selected healed lesions were biopsied to demonstrate tumor absence. Patients were selected for treatment if pretreatment testing of the tumor showed regression in vitro with heat exposure. Analysis of the early and midterm failures showed little sustained rise of the CD4+ cells if presenting total CD4+ counts were below 50 and had been at such low levels for extended periods, although other surrogate markers of HIV activity declined (semiquantitative PCR) during this period and is felt to support the hypothesis of apoptosis proposed in this illness. Analysis of the tumors of the few men not responding demonstrated elevated levels of IL-6 as compared to responders (12 vs < 1 pg/ml). At 120 days 29/31 patients remained alive (expected, 20). At 360 days, 21/31 remained alive (expected, 11). In no patient was HIV activity stimulated with heat exposure. CMV retinitis did clear in some patients treated (both techniques), but treatment alone did not prevent later development of retinopathy. EBV parameters were markedly altered in the short term with heat exposure in some patients. Few patients showed herpes simplex activation. Varicella-zoster virus remitted in some patients. There is utility in the use of systemic hyperthermia to control HIV and related malignancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Booster PCR: evaluation of an improved method for amplification of a few HIV-1 proviral DNA sequences.

A biphasic polymerase chain reaction designed as booster PCR for the detection of human immunodeficiency virus type-1 (HIV-1) was evaluated in samples containing a very low number of target DNA. We examined DNA samples obtained from chronically infected H9/HTLV-III B cells and purified plasmidic DNA containing the entire HIV-1 genome. By using booster PCR we detected HIV-1 DNA sequences up to 5 infected cells in samples containing about 2 micrograms of genomic DNA, and up to 1 copy of plasmidic DNA in samples containing about 0.5 microgram of genomic DNA. Otherwise by using standard PCR HIV-DNA up 100 infected cells and up to 20 copies from plasmidic DNA could be detected. Our experiments in amplification of HIV-1 proviral DNA have demonstrated that booster PCR enhances sensitivity of detection of standard PCR in small quantities of target sequences at least 20-fold with no loss of specificity.

Mice infection with HIV-1: a new mouse model for HIV-1 in vivo research.

In mice experimentally infected with 1 x 10(5) UI/mouse of HTLV-IIIB IgM antibodies were detected 10-12 days after the infection, reaching peak values two weeks later; the IgM seratiter progressively decreased thereafter and was negative at ten-eleven weeks. HIV p24 antigen was detected ten-fifteen days after infection and reached peak values five-six weeks later. Antigenemia subsequently decreased and showed an oscillating course with a progressive decrease which persisted throughout the observation period. Two weeks after infection we detected IgG antibodies to the major core protein p24; reactivity to gp41 was observed as early as reactivity to p24 and persisted throughout observation period. The IgG antibodies to all HIV epitopes peaked two-three weeks after infection; the time course showed a decrease after ten weeks, progressively decreasing thereafter. After sixty-five weeks of infection the IgG seratiter value was lower but remained positive. Viruses indistinguishable from HIV were isolated from the peripheral blood mononuclear cells of infected mice 30, 60, 180 days after infection. These seroimmunological and virological data confirm that the immunocompetent mouse may serve as a low-cost reproducible model for HIV-1 in vivo research.

Endocytosis constitute the infectious route of HIV-1 entry in human and rabbit monocytes lacking the CD4 receptor.

To obtain "functionally" CD4 negative human monocytes (0-5 CD4 +/1 x 10(6)/cells), 50 ng/5.10(5) cells of OKT4A were added daily after a pre-incubation with OKT4A (100 ng/5.10(5) cells. In our experimental conditions the blocking the CD4 receptor of human monocytes with OKT4A monoclonal antibody did not prevent HIV-1 infection, although the level of virus replication appeared lower than that in cultures without OKT4A. "Naturally"CD4 negative rabbit monocytes infected with HIV-1 also released a detectable level of virus after 12-15 up 28-30 days. In "naturally" CD4 negative rabbit monocytes and "functionally" CD4 negative human monocytes, the virus particles entering via phagocytosis are not infectious because multiple well defined virions were observed in phagocytic vacuoles and the envelopes of these particles did not appear to interact with the vacuolar membrane. The infectious particles were represented by endocytic vesicles containing only the core of HIV after fusion between the viral envelope and endocytic membrane. Fusion between the viral envelope and plasma membrane on the cellular surface was never observed, in spite of examining greater than 1000 virions bound the surface of human and rabbit macrophage monocytes. The absence of cytopathic effect in the rabbit and human CD4 negative monocytes infected with HIV-1, and conversely the presence of specific sequences of HIV in the genomic DNA may indicate that the macrophages-monocytes serve as an important reservoir for the persistence of HIV in infected hosts, similar to the other related Lentiviruses. Our virological data have also demonstrated that virus infection can be transmitted from rabbit and human infected monocytes to uninfected H9 cells. This preliminary study may offer important evidence for the development and testing of vaccines and compounds that inhibit HIV penetration of susceptible cells.

Kinetics of p24 antigenemia, IgM, IgG antibodies to p24 and p41 and Ig virus isolation in rabbits experimentally infected with HIV-1.

In rabbits experimentally infected with 1.10(5) u.i./ml HIV, IgM antibodies were detected 10-15 days after infection, reaching peak value two weeks later and remaining stable for two weeks long. Then a the IgM serotiters progressively decreased and were negative at ten weeks. HIV p24 antigen was detected ten-fifteen days after infection, reaching peak value five-six weeks later. Antigenemia subsequently decreased and reached a second peak after nine weeks. In our experimental conditions, the antigenemia persisted throughout the observation period. The IgG antibody titer reached a maximum two weeks after infection; the time course showed a decrease after ten weeks, followed by progressively decreasing fluctuating course. After twenty four weeks of infection the serotiter values though lower were always positive. Three-four weeks after infection we detected IgG antibodies to the major core protein p24. Reactivity of IgG antibodies to gp41 was observed earlier than reactivity to p24; these antibodies were detected over six months after infection. Viruses indistinguishable from HIV were isolated from the peripheral blood mononuclear cells of infected rabbits 30, 60 and 180 days after infection. These data further confirm that the rabbit may serve as an economical and reproducible model for HIV infection in which vaccines and antiviral agents could be tested.

Modification of norfloxacin inhibition of DNA gyrase induced by a 28 KDal DNA binding protein.

We have previously studied a clinical isolate of Providencia stuartii which showed high levels of resistance to 4-quinolones, aminoglycoside and beta-lactam antibiotics (Landini et al., 1987). DNA gyrase from this isolate was inhibited for 50% of activity at a concentration of 15 microM of norfloxacin, which is about 5-fold higher compared to the 50% inhibitory concentration for a standard DNA gyrase. It has been described that 4-quinolone inhibition of DNA gyrase is caused by their binding to DNA and by the distortion induced in DNA tertiary structure, and that affinity binding of 4-quinolones is different for DNAs in different structures. In order to detect whether the interaction between pAT 153 and a protein able to modify DNA tertiary structure could affect norfloxacin inhibitory concentrations for DNA gyrase we purified from the clinical isolate of Providencia stuartii a DNA binding protein of about 28 KDal which induces changes in supercoiling degree of DNA. Assays of DNA gyrase activity were performed on the complex pAT 153-DNA binding protein-norfloxacin. Results showed an increase from 15 microM to 20 microM of 50% norfloxacin inhibitory concentration for DNA gyrase when pAT 153 was complexed with the 28 KDal protein.

Detection of IgG anti-HIV antibodies in mice experimentally infected with HIV-1.

In the present work we report our preliminary data demonstrating the susceptibility of mouse to HIV infection. IgG antibodies were found in mice intraperitoneally inoculated with H9/HTLV-IIIB infected cells. Infected mice showed a humoral response and the antibodies detection includes specific immunoglobulin directed against the major gene product of HIV. Two weeks after infection with HIV infected cells we detected in mice IgG antibodies to p24, and the reactivity to gp41 was observed as early as reactivity to p24 and persisted throughout observation period. The persistence of specific antibody response to HIV after one year seems to demonstrate the possibility of the use of the mouse as animal model for HIV in vivo experiments. Studies concerning the degree and kinetic of HIV infection in mouse are in progress in our laboratory.

Infection of rabbits with human immunodeficiency virus.

An important requirement for the development of a vaccine against the human immunodeficiency virus (HIV-1), the causative agent of AIDS, is a readily available animal model that would allow possible immunogens to be evaluated. The only species to have been infected with HIV-1 so far is the chimpanzee. However, the scarcity of this animal and its designation as an endangered species place severe restrictions on its use as an animal model. Attempts to infect mice, rats, hamsters, guinea-pigs, musk shrews, and rabbits with HIV-1 or infected cells have all been unsuccessful. We now report that the intraperitoneal inoculation of rabbits with HIV-1 or chronically infected H9 cells consistently induces a persistent infection.

The morphogenesis of BLV (bovine leukemia virus) cultivated on FLK (fetal lamb kidney): an ultrastructural study.

A new cell line, obtained by co-cultivation of fetal lamb kidney cells and lymphocytes collected from an adult calf affected by enzootic bovine leukemia, was studied for bovine leukemia virus (BLV) morphogenesis. In this new cell line, called FLK-BLV, persistently infected with BLV, we identified extracellular and intracellular BLV particles. We never observed "budding" particles along the cell surface, and therefore assumed it was a new BLV maturation process in the cell vacuoles. In fact we found mature and non-mature particles connected with the cell-membrane system or cellular debris within vacuoles. We suggest that the viral envelope could be supplied by vacuole membrane. In our samples we also observed a cytopathic effect with syncytia formations similar to those observed on other BLV-producing cell lines.

Beta-lactam resistant Pseudomonas aeruginosa strains emerging during therapy: synergistic resistance mechanisms.

The emergence of beta-lactam resistant strains of Pseudomonas aeruginosa during treatment was studied. Three different strains present before treatment persisted with changes in their beta-lactam resistance during treatment. The isolates before and after therapy were studied for beta-lactamase production and permeability barrier. The increased beta-lactam resistance was correlated with an increased permeability barrier. In order to verify if the permeability barrier was correlated with changes in outer membrane proteins, outer membrane preparations were analyzed by SDS-PAGE. Several differences were observed between the OMP profiles of the post therapy and the pre therapy isolates. Furthermore, an analysis of PBPs pattern of strains studied was carried out and alterations in target proteins were observed.

Antiviral activity of Chamaecyparis lawsoniana extract: study with herpes simplex virus type 2.

Ethanolic extract of the green part of Chamaecyparis Lawsoniana was tested for antiviral activity and toxicity in tissue culture. All experiments were carried out in confluent human embryo lung fibroblasts. Treatment of fibroblast cells with extracts after viral inoculation was effective in inhibiting the replication of herpes simplex virus type 2 (HSV-2). The critical time for virus inhibition was 4 to 5 h after virus absorption. The antiviral activity was assayed employing the techniques of viral plaque and yield reduction. Toxicity of Chamaecyparis Lawsoniana in uninfected cells was studied as alteration of cell morphology, cellular viability and inhibition of host cell DNA synthesis. Herpes simplex virus inhibition occurred in presence of extract concentration of 0.5 micrograms/ml, whereas concentrations exceeding 5 micrograms/ml and 140 micrograms/ml were found to be cytotoxic when evaluated with inhibition of host DNA synthesis and cellular viability respectively. Results suggest that further investigations concerning the isolation of substances responsible for antiviral activity and an effort to define the mechanisms of action are warranted.

Modifications of DNA-gyrase and of permeability in a norfloxacin-resistant clinical isolate of Providencia stuartii.

We obtained a clinical isolate of Providencia stuartii showing a high level of resistance to norfloxacin and to other 4-quinolones, whose target is the enzyme DNA-gyrase. This strain showed resistance also to beta-lactam and aminoglycoside antibiotics. In order to detect modification of DNA-gyrase, we performed supercoiling assays in vitro in presence of norfloxacin and ciprofloxacin. Furthermore, outer membrane proteins, which are involved in permeability mechanisms, were analyzed on SDS-polyacrylamide gels. Results showed that both modifications in DNA-gyrase and changes in outer membrane proteins can be held responsible for resistance to 4-quinolones; moreover, these modification are probably supported by a third mechanism of resistance.

Preliminary characterization of an inhibitory activity of fetal bovine serum on the infectivity of rotavirus strain SA-11.

In the present work we studied non antibody inhibiting activity present in fetal bovine serum and active to Rotavirus infectivity and growth in cell cultures. This inhibitor was revealed by an in vitro neutralization test and characterized by gel filtration and chemical and enzymatic treatments. Furthermore, commercial preparations of bovine serum proteins were tested for inhibitory activity. Our results show that serum inhibition is partially resistant to trypsin and neuraminidase treatments but completely destroyed by KIO4. A similar activity was observed in a commercial serum bovine fraction containing predominantly alpha-globulins. These results seem to indicate that glycoproteins, and their glucidic components are the molecules predominantly involved in serum inhibition towards Rotavirus infectivity.

Human immunodeficiency virus (HIV): an ultrastructural study.

H-9 cells producing HIV were examined by electron microscopy to value the virus-host cell relationships. HIV fine structure was also studied. HIV induces little cellular damages and it can penetrate into the cytoplasm by phagocytosis. Phagocytosis of the virus could play an important role in the mechanism of cellular infection.

Occurrence of antipseudomonal beta-lactams and aminoglycosides resistance in Pseudomonas aeruginosa during therapy.

Four strains of Pseudomonas aeruginosa clinical isolates were studied for resistance to antipseudomonal beta-lactams and aminoglycosides. Two of these strains were isolated from two different patients before antibiotic treatment, the other two strains, isolated during therapy, developed resistance to many of antipseudomonal beta-lactams and, in addition, to aminoglycoside antibiotics. All the strains produced a constitutive chromosomal beta-lactamase, while the latter two showed a significant reduction in permeability coefficient. Thus, a permeability change may be the major factor involved in the resistance to most antipseudomonal beta-lactams and may be responsible for development of cross-resistance to aminoglycosides.