Extensive polymorphisms observed in HIV-1 clade B protease gene using high-density oligonucleotide arrays.

Naturally occurring mutations in HIV-1-infected patients have important implications for therapy and the outcome of clinical studies. However, little is known about the prevalence of mutations that confer resistance to HIV-1 protease inhibitors in isolates derived from patients naive for such inhibitors. In the first clinical application of high-density oligonucleotide array sequencing, the sequences of 167 viral isolates from 102 patients have been determined. The DNA sequence of USA HIV-1 clade B proteases was found to be extremely variable and 47.5% of the 99 amino acid positions varied. This level of amino acid diversity is greater than that previously known for all worldwide HIV-1 clades combined (40%). Many of the amino acid changes that are known to contribute to drug resistance occurred as natural polymorphisms in isolates from patients who had never received protease inhibitors.

Interferon: protection of cells infected with an intracellular protozoan (Toxoplasma gondii).

Chick and mouse cell monolayers treated with interferon were significantly protected from destruction by toxoplasma. The results suggest that fewer numbers of organisms were released from interferon-treated cells. Interferons were uniformly more active in a viral assay than in a toxoplasma assay, and they had the same properties in both assays. The data suggest that interferon activity may be directed against organisms phylogenetically higher than viruses.

[Interferon induction increased through chemical modification of a synthetic polyribonucleotide].

The alternating copolymer riboadenylic-ribouridylic acid gained asignificant increase in ability to stimulate interferon production (2-to 20-fold) and cellular resistance (100-to 10,000-fold) both in vitro and in vivo upon substitution of phosphate by thiophosphate groups. The resulting nucleotide analog was also 10 to 100 times less sensitive to degradation by pancreatic ribonuclease, as determined byresidual antiviral activity.

Interferon inducers in vitro: difference in sensitivity to inhbitiros of RNA and protein synthesis.

Interferon can be induced by diverse agents in a variety of mammalian cell cultures through apparently two mechanisms. One results in an early (2 to 10 hours) appearance of interferon and is relatively resistant to inhibition by actinomycin, puromycin, or fluorophenylalanine. A second mechanism results in a late (18 to 24 hours) appearance of interferon and is more sensitive to inhibition by these inhibitors. The molecular basis for each mechanism is unclear. Since each interferon inducer may have multiple effects on the cell, the differences observed may not necessarily reflect a fundamental difference in the mechanism of interferon stimulation.

Interferon, antibody, and other host factors in herpes zoster.

The influence of several factors on the course of herpes zoster was studied in 151 patients. Dissemination of zoster was associated with the presence of a concurrent disease, especially Hodgkin's disease, and/or the use of immunosuppressive therapy. Several host-immune parameters, including quantitative immunoglobulins, circulating lymphocyte counts, delayed hypersensitivity to multiple skin test antigens, and lymphocyte transformation to phytohemagglutinin did not correlate with dissemination of disease. Development of virus-specific complement-fixing antibody (CFA) was delayed in some patients with disseminated disease. Vesicle interferon (V-IF) titers were low early in the disease in patients with localized and disseminated zoster and then rose, usually abruptly, to a peak value and declined as pustulation and crusting occurred. However, titers in patients with localized disease rose at an earlier time. This could be seen in terms of time to development of intermediate values of V-IF or by the day on which the sharpest rise occurred. In 15 carefully studied patients with disseminated disease, the development of the maximum V-IF response was followed within 48 hr by cessation of dissemination. Half of the patients in this group had no CFA detectable until after dissemination had ceased. These findings suggest at least two host factors whose interaction might determine host response to zoster; local interferon production (possibly mediated by sensitized lymphocytes) and humoral antibody, acting to prevent or shorten dissemination of an initially local disease.

The interaction of human macrophages and lymphocytes in the phytohemagglutinin-stimulated production of interferon.

In studies of 13 normal adults to determine the blood cell types responsible for interferon production induced by phytohemagglutinin, the following observations were made. (a) In cultures containing 96-100% pure macrophages derived from blood monocytes, no interferon was detected in either the presence or the absence of phytohemagglutinin for up to 92 hr. (b) In cultures of 99.5-100% pure lymphocytes, low levels of interferon were detected in the presence, but not in the absence, of phytohemagglutinin. (c) An average fivefold increase in interferon titers occurred when pure lymphocytes were combined with the macrophages in culture with phytohemagglutinin. The peak response of interferon occurred at 68 hr after the initiation of the combined cultures. For maximum response, phytohemagglutinin was required for the duration of the culture, and both cell types in association were necessary. Medium from phytohemagglutinin-stimulated macrophages or lymphocytes could not substitute for the corresponding intact cell. However, frozen-thawed macrophages in combination with lymphocytes and phytohemagglutinin produced an intermediate interferon response. An increase in either cell type produced an increased response in the range studied: lymphocytes, 0.45-1.8 x 10(6) per ml; and macrophages, 0.5-2.1 x 10(5) per ml. Syngeneic fibroblasts, HeLa cells, or mouse macrophages could not substitute for the human macrophages in the combined cultures with phytohemagglutinin. (d) Although all cultures producing interferon showed some degree of transformation (thymidine-(3)H incorporation into deoxyribonucleic acid), no direct correlation between the degree of phytohemagglutinin-induced lymphocyte transformation and the interferon titers was observed.The demonstration of macrophage-lymphocyte interaction in the production of interferon is of interest in view of the known interrelationship of these same cell types in antibody synthesis and cellular immunity.

The role of interferon in the protective effect of a synthetic double-stranded polyribonucleotide against intranasal vesicular stomatitis virus challenge in mice.

Intravenous injection of polyinosinic acid/polycytidylic acid [(poly rI).(poly rC)] offered significant protection against intranasal challenge of young mice with vesicular stomatitis virus (VSV). Optimal protection was obtained when a single dose was administered 2 hr before virus challenge, but repeated doses were effective when started as late as 3 days after virus challenge. The therapeutic ratio or ratio of maximum tolerated dose to minimum effective dose for a single intravenous injection of (poly rI).(poly rC) 2 hr before virus inoculation was >/=8 mg/kg:0.004 mg/kg or >/=200.Dose-response curves for interferon production and antiviral protection by (poly rI).(poly rC) were closely parallel. Equivalent doses of poly rI or poly rC alone did not exert any interferon-inducing capacity or protective effect on intranasal VSV challenge. Several factors, which are known to potentiate or antagonize interferon production, increased or decreased the interferon-inducing capacity and antiviral protection of either (poly rI).(poly rC) or maleic acid/divinyl ether copolymer (MA/DVE) in parallel. Interferon production and antiviral protection by MA/DVE were enhanced by arginine but abolished by prior treatment with MA/DVE; DEAE-dextran (intraperitoneally), kinetin riboside and isopentenyladenosine, and prior injection of endotoxin reduced both interferon production and antiviral protection by (poly rI).(poly rC). Treatment with exogenous interferon in amounts which closely mimicked the levels of circulating interferon produced endogenously by an effective dose of (poly rI).(poly rC) gave protection against intranasal VSV which was identical with that dose of (poly rI).(poly rC). This strongly suggests that interferon production accounts for the whole protective effect of (poly rI).(poly rC) in the intranasal VSV assay.

Cellular and humoral immunity in the pathogenesis of recurrent herpes viral infections in patients with lymphoma.

86 patients with lymphoma were evaluated prospectively for clinical and laboratory evidence of recurrent varicella-zoster, herpes simplex, and cytomegalovirus infections during the first 16 mo of treatment. Cellular immunity to the viral antigens was measured by in vitro lymphocyte transformation and interferon production. Antibody titers and nonspecific measures of cellular immunity, including T-cell quantitation and transformation to phytohemagglutinin, were also assessed. The patients treated with radiation and chemotherapy had the highest incidence of reactivation of each of the viruses (15-19%). Greater susceptibility to herpes viral reactivation in these patients correlated with suppression of cell-mediated immunity to the specific virus. In individual patients, suppression of cellular immunity to the specific herpes viral antigen preceded each episode of reactivation, but recurrent infection did not occur in all patients with diminished specific lymphocyte transformation. Absence of the response appears to be a necessary but not a sufficient condition for the recrudescence of latent infection. Better preservation of cellular immunity to herpes simplex antigen during treatment was associated with infrequent reactivation of herpes simplex. In 25 patients with acute herpes zoster, uncomplicated recovery from the infection was accompanied by the development of lymphocyte transformation and interferon production to varicella-zoster antigen. Quantitation of T-cell numbers and phytohemagglutinin transformation did not correlate with the presence of viral cellular immunity in treated patients. Responses returned while T-cell numbers were low, and the recovery of phytohemagglutinin transformation often preceded recovery of the responses to viral antigens. Although some patients had deficiencies in viral cellular immunity at diagnosis, the duration of the suppression of specific antiviral responses resulting from treatment appears to be the most important factor predisposing to the recurrence of herpes infections in lymphoma patients.

Inactivation of S-adenosylhomocysteine hydrolase during adenine arabinoside therapy.

To assess for possible inhibition of cellular transmethylation during adenine arabinoside (ara-A) therapy, S-adenosylhomocysteine hydrolase activity was analyzed in 10 patients with chronic hepatitis B virus infection. In six patients receiving ara-A, enzyme activity was suppressed to 0-2% of control erythrocyte enzyme activity. This decrease in enzyme activity was evident within 4 h of starting the drug infusion and continued for 7 d after cessation of therapy. S-adenosylhomocysteine hydrolase activity of peripheral mononuclear cells was also measured in two patients receiving ara-A. Suppression to as low as 3.5% of pretreatment levels was found; however, marked fluctuations with partial return of enzyme activity during therapy was also observed in mononuclear cells. Inhibition of an enzyme involved in transmethylation reactions was observed in patients during ara-A therapy. This could contribute to the side effects and antiviral properties of ara-A.

Reduction in plasma human immunodeficiency virus ribonucleic acid after dideoxynucleoside therapy as determined by the polymerase chain reaction.

Cell-free HIV RNA in plasma was detected and quantitated after antiviral therapy by the polymerase chain reaction. RNA was extracted from plasma, reverse transcribed to cDNA, amplified by polymerase chain reaction, and quantitated by absorbance based on an enzyme-linked affinity assay. 72 HIV antibody-positive subjects had one plasma sample taken. 39 who were not receiving antiretroviral therapy at the time had a mean plasma HIV RNA copy number of 690 +/- 360 (mean +/- SEM) per 200 microliters of plasma, while 33 subjects who had been receiving zidovudine therapy for a minimum of 3 mo had a mean copy number of 134 +/- 219 (P less than 0.05). 27 additional HIV antibody-positive patients had two plasma samples taken before and 1 mo after initiating dideoxynucleoside therapy. Plasma HIV RNA copy number fell from 540 +/- 175 to 77 +/- 35 (P less than 0.05). Finally, nine of these subjects had two baseline samples obtained before initiating therapy and two posttreatment samples 1 and 2 mo after therapy was begun. Mean plasma RNA copy number declined from 794 +/- 274 to less than 40 (below the lower limit of sensitivity) after 1 mo of therapy, with suppression maintained after 2 mo of therapy. These results suggest that gene amplification can be used to detect and quantitate changes in plasma HIV RNA after dideoxynucleoside therapy. Plasma HIV polymerase chain reaction may be a more sensitive marker to monitor antiviral therapy, particularly in asymptomatic patients where measurement of p24 antigen or quantitative plasma cultures are negative.

Evolution of recurrent herpes simplex lesions. An immunohistologic study.

We performed immunoperoxidase stains on skin biopsies taken from nine patients with recurrent peripheral herpes simplex lesions at 12 h to 6 d after onset of signs of symptoms to phenotype the inflammatory infiltrate, to detect cells producing interferons alpha and gamma, and to locate herpes simplex virus antigen-containing cells. Viral glycoprotein antigen was located in the nuclei and cytoplasm of necrotic epidermal cells, often within vesicles, in biopsies taken between the first and third day. Histologically, biopsies of all stages showed intradermal focal perivascular and diffuse mononuclear inflammatory infiltrates. The cells constituting the infiltrates were predominantly T lymphocytes with lesser numbers of histiocytes; Leu 7+ (most natural killer/killer) cells and B cells were rare in the biopsy specimens. Leu 3a+ ("helper") T lymphocytes predominated in both subepidermal and perivascular regions of early lesions (12-24 h). Tissue helper/suppressor ratios ranged from 6.3 to 3.4 compared with 1.9-1.0 in blood. In later lesions (after 2 d), monocytes/macrophages were more prominent in tissue sections and the helper/suppressor ratios (2.3-2.5) more nearly approximated those of blood (1.6-2.7). The negative correlation of tissue ratios with time was significant (P less than or equal to 0.02). A large proportion of the infiltrated T lymphocytes expressed DR antigens. There was also diffuse strong DR expression on epidermal cells in five cases (all of two or more days). In six biopsies, scattered macrophages and small cells, presumably lymphocytes, demonstrated cytoplasmic or membrane staining for a substance which copurifies with interferon gamma. We identified such stained cells within vessels, suggesting that these cells circulate. Gamma interferon might have an important role within the herpetic lesions, possibly inducing macrophage activation and cytotoxic T lymphocytes and increasing DR expression on monocyte and epidermal cells.

A 6-basepair insert in the reverse transcriptase gene of human immunodeficiency virus type 1 confers resistance to multiple nucleoside inhibitors.

While many point mutations in the HIV-1 reverse transcriptase (RT) confer resistance to antiretroviral drugs, inserts or deletions in this gene have not been previously characterized. In this report, 14 RT inhibitor-treated patients were found to have HIV-1 strains possessing a 6-basepair insert between codons 69 and 70 of the RT gene. Known drug resistance mutations were also observed in these strains, with T215Y appearing in all strains. Genotypic analysis indicated that the inserts had substantial nucleotide variability that resulted in relatively restricted sets of amino acid sequences. Linkage of patients' treatment histories with longitudinal sequencing data showed that insert strains appeared during drug regimens containing ddI or ddC, with prior or concurrent AZT treatment. Drug susceptibility tests of recombinant patient isolates showed reduced susceptibility to nearly all nucleoside RT inhibitors. Site- directed mutagenesis studies confirmed the role of the inserts alone in conferring reduced susceptibility to most RT inhibitors. The addition of AZT-associated drug resistance mutations further increased the range and magnitude of resistance. These results establish that inserts, like point mutations, are selected in vivo during antiretroviral therapy and provide resistance to multiple nucleoside analogs.

Growth regulation of melanoma cells by interferon and (2'-5')oligoadenylate synthetase.

We report that endogenous, as well as exogenous, interferon (IFN) regulates the growth of human melanoma cells in culture. When antibodies directed against human fibroblast IFN were incorporated into the media of high-density cells stimulated to proliferate with serum, the cells entered the cell cycle earlier than did the controls. In investigating the biochemical basis for this finding, we have found that there is an inverse relationship between the (2'-5')oligoadenylate synthetase levels and the percentage of cells in S in untreated cultures. Upon IFN treatment, the relationship is obliterated and (2'-5')oligoadenylate synthetase levels increase throughout all phases of the cell cycle. This increase in enzyme levels correlates well with the decreased probability of the IFN-treated cells to cycle. These findings suggest a biological role for IFN as a negative growth factor for cells in culture.

Comparison of combinations of interferons with tumor specific and nonspecific monoclonal antibodies as therapy for murine B- and T-cell lymphomas.

Two murine models, C3H 38C13 B-cell lymphoma and AKR SL2 T-cell lymphoma were used to determine the efficacy of three different interferon preparations, recombinant human hybrid interferon-alpha A/D, recombinant murine interferon (rMIFN)-gamma, and natural MIFN-alpha/beta (greater than or equal to 85% beta), alone and in combination with tumor specific and nonspecific monoclonal antibody therapy. All three interferon preparations have direct in vitro antigrowth activity for 38C13 and SL2. All three interferons have direct antitumor activity in vivo for 38C13 lymphoma at high doses; however, none of these interferons has independent antitumor activity for SL2 in vivo. These data indicate that there is no relationship between in vitro growth cytostasis/cytolysis and in vivo antitumor response. All three interferon preparations will potentiate both tumor specific and nonspecific monoclonal antibody therapy. Natural MIFN-alpha/beta and recombinant human hybrid interferon-alpha A/D, which should share a common cell surface receptor, had similar antitumor activity in both models. Combining recombinant human hybrid interferon-alpha A/D and rMIFN-gamma therapy was not additive for 38C13 lymphoma and a three-way combination with antiidiotype was not significantly more effective than combination therapy with one interferon type. In general, rMIFN-gamma was more effective in in vivo combination therapy against the s.c. T-cell lymphoma than against the i.p. B-cell lymphoma and was more synergistic with anti-Thy1.1 than with antiidiotype.

Enhanced in vivo therapeutic response to interferon in mice with an in vitro interferon-resistant B-cell lymphoma.

A stable subline of 38C13 B-cell lymphoma (SIR-1) resistant to the antiproliferative effects of alpha-interferon (IFN) was isolated. In addition to defects in antiproliferative effects of IFN, SIR-1 is defective in IFN-mediated antiviral activity against both encephalomyocarditis virus and vesicularstomatitis virus. It is also defective in the induction of 2'-5'-oligoadenylate synthetase mRNA and enzyme activity, enhancement of H-2 antigen expression, and transient induction and subsequent repression of c-myc by IFN. SIR-1, although completely resistant to IFN in vitro, is more sensitive to IFN than the parental cell line in vivo. IFN treatment at 10(4) units, three times weekly, resulted in a 28% increase in mean survival time and a 1.4% long term survival rate in the IFN-sensitive 38C13 cell line but resulted in a 275% increase in mean survival rate and a 27% long term survival rate in the interferon-resistant SIR-1 mutant. Statistical analysis of 38C13 and SIR-1 with and without IFN treatment demonstrate that: a) the SIR-1 mutant remains sensitive to the cytotoxic effects of IFN in vivo (P less than 0.0001); and b) the mean survival and long term survival of animals with the SIR-1 mutant is significantly greater than for animals with the IFN-sensitive 38C13 cell line (P less than 0.0001). Two additional independently isolated IFN-resistant cell lines (SIR-111 and SIR-E102) also demonstrate significantly enhanced in vivo response to IFN compared to the interferon-sensitive parental (38C13) cells. These results indicate that, for this cell line, the antitumor effects of IFN are mediated by activation of host defenses and that resistance to the in vitro cytotoxic effects of IFN results in a tumor phenotype that is more readily recognized by host defenses and eliminated.

Impact of prophylactic immediate posttransplant ganciclovir on development of transplant atherosclerosis: a post hoc analysis of a randomized, placebo-controlled study.

BACKGROUND: Coronary artery disease occurs in an accelerated fashion in the donor heart after heart transplantation (TxCAD), but the cause is poorly understood. The risk of developing TxCAD is increased by cytomegalovirus (CMV) infection and decreased by use of calcium blockers. Our group observed that prophylactic administration of ganciclovir early after heart transplantation inhibited CMV illness, and we now propose to determine whether this therapy also prevents TxCAD. METHODS AND RESULTS: One hundred forty-nine consecutive patients (131 men and 18 women aged 48+/-13 years) were randomized to receive either ganciclovir or placebo during the initial 28 days after heart transplantation. Immunosuppression consisted of muromonab-CD3 (OKT-3) prophylaxis and maintenance with cyclosporine, prednisone, and azathioprine. Mean follow-up time was 4.7+/-1.3 years. In a post hoc analysis of this trial designed to assess efficacy of ganciclovir for prevention of CMV disease, we compared the actuarial incidence of TxCAD, defined by annual angiography as the presence of any stenosis. Because calcium blockers have been shown to prevent TxCAD, we analyzed the results by stratifying patients according to use of calcium blockers. TxCAD could not be evaluated in 28 patients because of early death or limited follow-up. Among the evaluable patients, actuarial incidence of TxCAD at follow-up (mean, 4.7 years) in ganciclovir-treated patients (n=62) compared with placebo (n=59) was 43+/-8% versus 60+/-10% (P<0.1). By Cox multivariate analysis, independent predictors of TxCAD were donor age >40 years (relative risk, 2.7; CI, 1.3 to 5.5; P<0.01) and no ganciclovir (relative risk, 2.1; CI, 1.1 to 5.3; P=0.04). Stratification on the basis of calcium blocker use revealed differences in TxCAD incidence when ganciclovir and placebo were compared: no calcium blockers (n=53), 32+/-11% (n=28) for ganciclovir versus 62+/-16% (n=25) for placebo (P<0.03); calcium blockers (n=68), 50+/-14% (n=33) for ganciclovir versus 45+/-12% (n=35) for placebo (P=NS). CONCLUSIONS: TxCAD incidence appears to be lower in patients treated with ganciclovir who are not treated with calcium blockers. Given the limitations imposed by post hoc analysis, a randomized clinical trial is required to address this issue.

Treatment of nasopharyngeal carcinoma with human leukocyte interferon.

Nasopharyngeal carcinoma (NPC) is a human neoplasm closely associated with Epstein-Barr virus (EBV). Human leukocyte interferon (IFN) has known antiviral and antineoplastic properties. After initial IFN treatment in one NPC patient demonstrated acceptably low toxicity, 12 additional patients were treated on a protocol with IFN, 10 X 10(6) units intramuscularly (IM) daily for 30 days. IFN did not affect serum anti-EBV antibody titers (IgA and IgG antiviral capsid and early antigens). Of six patients tested, none was found to excrete EBV in saliva before, during, or after IFN. Four patients had measurable tumor regression (two partial responses and two minor responses), three had stable disease, and five patients plus the initial preprotocol patient had progressive disease. Toxicity included fever, fatigue, and myalgias in all patients, thrombocytopenia in two patients, and neutropenia in three patients. Three patients were withdrawn from the study, one each for severe fatigue, neutropenia, and hypotension. This study demonstrates that IFN has sufficient activity in advanced NPC to justify further investigation.

Nonviral interferon inducers.

Interferon production can be stimulated by a great variety of microbial and nonmicrobial agents other than viruses. The nonmicrobial inducers can be divided into polyanions, mitogens, and a miscellaneous category including the various endotoxins and antibiotics. The polyanions appear to require a stable, high molecular weight backbone and a high density of free anionic groups whether they are polynucleotides, plastics, or polysaccharides. Mitogen-induced interferon appears to be but one of a constellation of substances produced following lymphocyte transformation. The process of transformation can be stimulated either by specific immune recognition or non-specifically by phytohemagglutinin. Synthetic polynucleotide inducers are active; the thermostable, double-stranded RNA's are much more active than the double-stranded DNA's or 1-, 3-, or 4-stranded RNA's. Some success has been obtained with potentiation of nucleotide inducers through the use of polycationic substances, complexing with a polysaccharide, concurrent administration of a metabolic antagonist, or substitution of phosphate by thiophosphate in the polynucleotide backbone. The stages in the interaction of interferon stimulating RNA and cells can be divided into three steps: first, binding to cell surface, next, a temperature dependent "recognition" step, and finally, degradation and utilization of monomers in cellular RNA synthesis; the critical recognition site has not yet been determined. The vast majority of cell-associated polynucleotide remains at the surface of the cell. Information from animal models resembling human diseases suggests that certain of these nucleotide inducers may have clinical usefulness in therapy or prophylaxis.

Zoster immune globulin prophylaxis of disseminated zoster in compromised hosts. A randomized trial.

Herpes zoster can be a severe, and sometimes fatal, virus infection in its disseminated form in immunocompromised hosts. Previous studies have suggested that delay in appearance of antibody to varicella-zoster virus occurs as one defect in such patients. In this study, pooled gamma-globulin (normal serum globulin [NSG]) and zoster immune globulin ([ZIG] prepared from convalescent zoster patients) were compared for their ability to prevent dissemination of early localized zoster in immunocompromised hosts. Either agent was given intramuscularly in randomized double-blind fashion within nine days of onset of zoster in 97 patients. Despite greater than 100-fold differences in titer of anti-varicella-zoster virus antibody, ZIG did not appear superior to NSG in prophylaxis of dissemination or diminishing postherpetic pain in zoster in immunocompromised hosts. Zoster immune globulins should be reserved for prophylaxis and modification of varicella, where its beneficial effect has been demonstrated.

Suppressive effect of interferon on erythroid cell proliferation.

The effect of interferon on mouse and human in vitro erythropoiesis was investigated using the mouse marrow CFU-E and the human blood BFU-E systems, respectively. Homologous interferons were found to have a marked inhibitory effect on mouse CFU-E and human BFU-E proliferation. This inhibitory effect was dose related and independent of the erythropoietin concentration used. The effective concentration of human interferon was within the range observed in humans following viral infections. It is suggested that interferon may play a role in the mechanism of the acute erythroblastopenic crisis observed at times in patients with chronic anemia following viral infections.