Viral infection and host defense.

Double-stranded RNA, made as an intermediary substance in the replication of most, if not all, viruses, may play a much more important role in the pathogenesis and the recovery from virus infections than has hitherto been suspected. Apparently, dsRNA is used by both the challenge virus and the host cell in an attempt to gain "molecular control." Double-stranded RNA exerts a set of effects, which may be well balanced, not only at the level of the individual cell but also at the complex assemblage of these cells termed the organism (Fig. 1). In the cell, interferon synthesis is triggered, although interferon mRNA translation may not occur if dsRNA shuts off protein synthesis too quickly. In the whole organism, the disease severity will depend on how certain toxic reactions evoked by infection (such as cell necrosis and fever) are counterbalanced by an increase in the host defense mechanisms (for example, immune responsiveness and interferon production). Many aspects of the response, relating to either progress of, or recovery from, the disease, can be explained on the basis of a dsRNA. In addition to drawing attention to the biodynamic role of dsRNA, our hypothesis suggests specific experimental vectors designed to enhance our information on the molecular basis of the morbid process which occurs with viral infection. Finally, we suggest that, although the dsRNA molecule may be viewed as a rather simple unit structure, the opportunity for further diversity in the biological activity of a given dsRNA molecule always exists. Namely, each deviation from a perfectly double-helical arrangement introduces the possibility for emphasizing one biological reactivity at the expense of another. This latter structure-activity property may partially account for the extreme apparent diversity, commonly encountered, in the presentations of virologic illness. Appendix note added in proof. Subsequent to submission of this text, we have found that the potent mitogen effect of dsRNA for lymphocytes (murine and human) is also exquisitively sensitive to the fidelity in base pairing of the input polymer pair (59). For example, infrequent "loops" (one nucleotide per 20 base pairs) in an otherwise perfectly helical rI(n) (.) rC(n) molecule [for example, rI(n) (.) r(C(19,)U)(n)] strongly changes its mitogenic properties. This observation, which supports our thesis that a "fine structure" term can be developed for other reactions triggered by dsRNA's in biological systems, emphasizes that diverse biological effects may be encountered with an ostensibly uniform family of dsRNA's.

Ribosomes: effect of interferon on their interaction with rapidly labeled cellular and viral RNA's.

Rapidly labeled RNA of mouse L cells and labeled RNA of Mengo virus, unlike cellular RNA labeled under steady-state conditions, form detectable complexes with L-cell ribosomes. These ribosome-RNA complexes formed in vitro appear analogous to those assembled during polysome formation in vivo. When ribosomes are prepared from L cells exposed to homologous interferon, their capacity to associate with cell messenger is preserved, while their ability to interact with viral RNA is markedly reduced. The ribosomes from cells exposed to interferon are thus altered selectively to permit only certain messages to be bound and translated.

Noncycling tumor cells are sensitive targets for the antiproliferative activity of human interferon.

Resting Burkitt's lymphoma cells (Daudi) in culture are more sensitive targets for the antiproliferative activity of purified human fibroblast interferon than cells that are rapidly multiplying. Thus, interferon may be of significant clinical value in neoplasms involving stem cells and, after chemotherapy, in suppressing the reemergence of tumors.

Human brain tumor--derived cell lines: growth rate reduced by human fibroblast interferon.

The biological response modifier human beta-interferon had pronounced antigrowth effects on various histologic types of human brain tumor cells but no effects on a nontransformed cell line, MRC-5. The cultures of brain tumor cells showed severe alterations indicative of cell injury and death after exposure to beta-interferon for 2 to 6 days. Similar results were obtained with cells freshly explanted from human brain tumors. The results indicate that it may be possible to use fresh, explanted tumor tissue to identify patients who might benefit from therapy with beta-interferon.

Friend leukemia: rapid development of erythropoietin-independent hematopoietic precursors.

Infection of mice with the polycythemia-inducing strain of Friend leukemia virus caused a rapid emergence of new erythroid precursor cells. These cells which, in the absence of erythropoietin, proliferated in vitro to form colonies and even differnetiated, quickly out-numbered the usual erythropoietin-dependent hematopoietic elements in bone marrow and spleen. Ultimately, the marrow and spleen were probably totally repopulated with this erythropoietin-independent cell.

Antiproliferative activity of highly purified mouse interferon: brief communication.

The antiproliferative effect of mouse L-cell interferon, purified by bovine serum albumin--agarose chromatography (which yields a sp act of 3 X 10(8) international reference U/ml protein), and its two subcomponents, obtained by CH-Sepharose 4B chromatography, has been studied in Balb/3T3 fibroblast cultues. Four to 10 international reference U of interferon/ml resulted in a 50% inhibition of DNA synthesis and cell growth, as determined by the incorporation of [3H]thymidine and cell counts, respectively. Hence the degree of inhibition produced by the purified interferon preparations was comparable to that produced by the original crude interferon (sp act, 1.5 X 10(6) international reference U/mg protein). Our results, obtained with L-cell mouse interferon purified by two novel affinity ligands, provided further evidence linking the antiviral and antiproliferative activities of this molecule.

Bypassing the "species barrier" with carbohydrate-altered interferon from leukocytes.

Interferon, a glycoprotein with demonstrated antitumor and antiviral properties, can be obtained from cells of different species, and some of these interferons can be effective across species lines. I hypothesize that the ability to cross or not to cross species lines lies in the carbohydrate moiety, and cross-species biological activity is a property of the polypeptide. This would represent one of the first major roles uncovered for glycosylation in any biological system. If this hypothesis is correct, then it should be possible to use animal-derived interferon as a large scale, inexpensive source to treat human diseases.

Antiproliferative and immunomodulatory actions of beta-interferon and double-stranded RNA, individually and in combination, on human bladder tumor xenografts in nude mice.

A cell line, RT4, derived from a human transitional cell carcinoma of the bladder, was grown as a xenograft in athymic mice. The growth of the xenografts was inhibited by beta-interferon (IFN-beta), polyriboinosinic acid.polyribocytidilic acid, the mismatched double-stranded RNA analogue r(I)n . r(C12,U)n, and to a lesser extent recombinant IFN-beta, when treatment was initiated at the time of tumor inoculation. In contrast, the growth rate of established tumors, approximately 6 mm in diameter at the initiation of therapy, was inhibited by both double-stranded RNAs, but not natural IFN-beta, indicating a possible tumor size dependence on the effectiveness of IFN-beta. Combinations of natural or recombinant IFN-beta with either polyriboinosinic acid.polyribocytidilic acid or r(I)n.r(C12,U)n gave an antagonistic effect regardless of tumor mass at the initiation of treatment. This antagonism could be overcome by alternating r(I)n. r(C12,U)n and natural IFN-beta treatment. Natural killer cell activity against RT4 cells in culture was augmented in the spleens of mice treated with r(I)n.r(C12,U)n, but not in those treated with natural IFN-beta. RT4 cells treated in culture with IFN-beta, however, were significantly less efficient as targets for natural killer cells from r(I)n.r(C12,U)n-treated and control spleens. These results indicate that: the effectiveness of IFN-beta may be related to the tumor mass; double-stranded RNAs appear to work, at least partially, in an indirect, immunomodulatory manner; combination therapy can yield an antagonistic rather than an additive or synergistic antitumor effect; and strategic scheduling can overcome the antagonistic effect of combination therapy.

Low natural cytotoxicity of peripheral blood mononuclear cells in individuals with high familial incidences of cancer.

The possible role that natural cell-mediated cytotoxicity may play as a host defense mechanism against malignant tumors was investigated. We measured natural cell-mediated cytotoxicity (51Chromium released) in 79 normal individuals using K562 leukemia cells as targets in quadruplicate assays after 3, 4, and 5 hr of incubation using three different effector:target cell ratios (6.2:1, 25:1, and 50:1). Natural cell-mediated cytotoxicity was significantly lower (p less than 0.005) in each of the nine separate assay conditions for individuals with a high familial incidence of cancer compared to individuals with a low incidence of cancer. Moreover, natural cell-mediated cytotoxicity inversely correlated with the number of family members with cancer. The relationship between high familial cancer incidence and low natural cell-mediated cytotoxicity was observed in males as well as in females and in nonsmokers as well as in smokers. The same conclusion was reached whether the data were expressed as percentage of 51Chromium released, as lytic units per 10(7) mononuclear cells, or as lytic units per ml of peripheral blood. Thus, defects in natural cell-mediated cytotoxicity may play a role in the initial stages of human tumorigenesis. It may also be possible to identify individuals at increased risk of cancer development.

Fibroblast interferon treatment of a patient with chronic active hepatitis. Increased number of circulating T lymphocytes and elimination of rosette-inhibitory factor.

A 23 year old woman with chronic active hepatitis documented by liver biopsy demonstrated persistent hepatitis B surface antigen, hepatitis B virus specific DNA polymerase hepatitis B core antigen (HBcAg), for approximately one year. The number of circulating T lymphocytes that rosetted with sheep erythrocytes was decreased, and a rosette-inhibitory factor was present in her peripheral blood. Interferon treatment (1 X 10(6) U/day intramuscularly for 82 days) resulted in a decrease of HBsAg and disappearance of HBcAg, (HBeAg) and specific DNA polymerase. In addition, the number of T lymphocytes increased to normal, and the rosette-inhibitory factor disappeared from the circulation. These findings suggest that the effect of interferon in chronic active hepatitis is mediated in part through its action on the immune system.

Purified human fibroblast interferon in vivo: skin reactions and effect on bone marrow precursor cells.

Human interferon from normal diploid fibroblasts, purified by sequential chromatography on concanavalin A-agarose and phenyl-sepharose, was administered parenterally in 4 subjects. Fever, marked skin hypersensitivity reactions and suppression of marrow stem cells (estimated by the count of myeloid colony-forming cells), side-effects common for less purified fibroblast and leukocyte interferons, were absent. Purified fibroblast interferon retained antiviral and immunomodulatory activity, evidenced by reduction of the blastogenic response of peripheral lymphocytes and decrease of hepatitis B virus markers in a patient with chronic hepatitis B infection treated with this substance.

Differential effects of human natural interferon-alpha and mismatched double-stranded RNA against a human renal cell carcinoma xenograft.

The antitumor effects of natural human IFN-alpha and mismatched dsRNA against the human renal cell carcinoma cell line 786-0 were studied both in a clonogenic soft agar assay and in the nude mouse. The 786-0 cells were sensitive in vitro to the antiproliferative effects of IFN-alpha in a dose-response manner, up to 3000 IRU/ml. These cells were also sensitive, in a dose-dependent manner, to mismatched dsRNA in the clonogenic assay. Mismatched dsRNA was effective in inhibiting tumor growth (p less than 0.001) in nude mouse xenografts, with regression of the tumor mass seen in all animals. A significant increase in survival (p less than 0.001) was seen in the mismatched dsRNA treated group. In contrast, IFN-alpha did not inhibit tumor growth in vivo, even though significant titers of IFN-alpha (greater than 3,000 IRU/ml) were found in the serum shortly after treatment. Mismatched dsRNA did not induce the production of human IFNs by the tumor cells in vitro. Assays of mouse IFN induction and their in vitro antigrowth effects indicated that the in vivo antiproliferative effect of mismatched dsRNA was probably not due to potentiation of any direct effects by the induced mouse IFNs. Tumor growth inhibition appeared to occur, at least in part, from the significant augmentation (p less than 0.01) of natural killer cell activity by mismatched dsRNA, as measured in the spleen cells of treated mice. These results suggest that, although both IFN-alpha and mismatched dsRNA can be directly antiproliferative against this tumor, either the IFN-independent antitumor effects of mismatched dsRNA or the mismatched dsRNA-induced augmentation of the host immune response plays a major role in tumor regression. Potentially, both mechanisms may be important in this system.

Rapid improvement of acute pulmonary edema with sublingual captopril.

OBJECTIVE: To test the hypothesis that sublingual captopril produces a more rapid improvement of acute pulmonary edema (APE) than does placebo, when added to a standard regimen of O2, nitrates, morphine, and furosemide. METHODS: Prospective, randomized, double-blind, placebo-controlled clinical trial in an urban teaching hospital ED. Adults brought to the ED with APE were given captopril or placebo sublingually. Every 5 minutes a clinical APE distress score (APEX) was obtained. RESULTS: Over the first 40 minutes of treatment, the mean APEXs were significantly better for the patients given captopril [p < 0.001, F = 14.5, one-way (repeated-measures) analysis of variance (ANOVA)]. At 30 minutes, the patients given captopril had a mean APEX improvement of 43% (i.e., to 57% of initial distress); the group given the current standard regimen plus placebo improved only 25% (i.e., to 75% of initial distress; p = 0.03, multiway ANOVA). In addition, there was less respiratory failure necessitating mechanical ventilation in the captopril patients (9%) vs the placebo patients (20%), which did not achieve significance (p = 0.10, Fisher's exact test). CONCLUSION: In APE, the addition of sublingual captopril to the standard regimen of O2, nitrates, morphine, and furosemide produces more rapid clinical improvement than does the standard regimen alone.

Declining rate of cricothyrotomy in trauma patients with an emergency medicine residency: implications for skills training.

OBJECTIVE: To report the change in cricothyrotomy rate with emergency medicine (EM) residency development and to address the implications for training in this skill. METHODS: A retrospective chart review was used to determine the cricothyrotomy rate at a 1,000-bed urban Level-1 trauma center with EM, surgery, and anesthesiology residencies. All adult trauma patient visits to the ED between July 1, 1985, and June 30, 1995, were reviewed. The cricothyrotomy rate was defined as the total number of cricothyrotomies per trauma admissions during a study phase. RESULTS: The study period was divided into 3 phases. Phase 1 (academic years 1985-1989): prior to the inception of the EM residency; phase 2 (academic years 1990-1992): initiation and establishment of the residency; and phase 3 (academic years 1993-1994): full implementation of the EM residency. The cricothyrotoiny rate during phase 1 was 1.8% (95% CI: 1.6 to 2.0), vs 1.1% (95% CI: 0.0 to 2.8) and 0.2% (95% CI: 0.0 to 0.2) during phases 2 and 3, respectively. CONCLUSIONS: The cricothyrotomy rate decreased with the full implementation of the EM residency. Whether this trend was an effect of the presence of an EM faculty and residency training program, a parallel approach to airway management nationwide, or another unidentified factor will require further investigation. Nonetheless, given the increasing rarity of this procedure, it is likely that many EM, surgical, and anesthesiology residents will not acquire clinical experience with this technique during training.

Occupational exposures to blood among emergency medicine residents.

OBJECTIVES: To investigate the epidemiologic characteristics of potentially infectious occupational exposures to blood among emergency medicine (EM) residents. METHODS: A SAEM-sponsored multiple-choice survey was administered anonymously to all EM residents participating in the 1998 American Board of Emergency Medicine in-service examination. Survey questions included resident demographics, use of universal precautions, frequency and types of exposures to blood, and exposure reporting. Residents who experienced at least one exposure were then asked to complete an additional set of questions referring only to their latest exposure. Mean values were calculated for each variable and differences between groups were compared by chi-square analysis. RESULTS: Three thousand one hundred sixty-two surveys were distributed to the resident participants, and 2,985 surveys (94.4%) were returned. Of the participants, 56.1% reported at least one exposure to blood during their EM training. The frequency of this self-reported exposure increased with advancing EM level of training (43% EM-1, 58% EM-2, 64% EM-3, 76% EM-4, p<0.001). Of these residents, 36.6% always followed universal precautions, 54% frequently, and 9.4% sometimes, rarely, or never. Those individuals who "always" followed universal precautions reported significantly fewer exposures than those who did not (p<0.005). The latest exposures were most commonly caused by a solid needle or sharp object (39.4%), by a hollow-bore needle (30.6%), or by eye splashes (17.2%). Of these exposures, 71.7% occurred in the ED setting, and only 46.7% of these exposures were reported to health care providers. CONCLUSION: Emergency medicine residents are frequently exposed to blood, most commonly due to puncture injuries by sharp objects. The rate of exposure reporting is low, which may compromise appropriate postexposure counseling and prophylaxis.

Tuberculosis exposure risk in emergency medicine residents.

OBJECTIVES: To assess purified protein derivative (PPD) test surveillance and respiratory protection practices of emergency medicine (EM) residents, along with the prevalence of PPD test conversion and the development of active tuberculosis (TB) in EM residents. METHODS: The study instrument was an anonymous, self-reporting, multiple-choice survey administered to U.S. and Canadian EM residents. It was distributed for voluntary completion in conjunction with the American Board of Emergency Medicine's annual in-service examination, which was administered February 25, 1998. RESULTS: A total of 89.3% (n = 2,985) of residents eligible to complete the survey completed at least part of it. The majority of residents are PPD-tested once a year. The prevalence of PPD test conversions in EM residents was between 1.4% (36/2,575) and 2.0% (52/2,575). Of the residents who PPD test-converted, the ED was most often the perceived area of TB source exposure (n = 15). Two residents (0.08%) reported having developed active TB, including chest radiographic findings or clinical infection, which equals a 0.14% (95% CI = 0.005 to 0.31) risk of developing active TB over a three-year residency. Half of all the residents do not routinely wear National Institute for Occupational Safety and Health (NIOSH)-approved particulate filtration respirator (PFR) masks in patient encounters at risk for TB exposure. While more than a third of EM residents have not undergone fit testing for a NIOSH-approved PFR mask, the lack of routine easy availability of such masks is the most common reason they are not routinely worn by EM residents during at-risk encounters for TB transmission. CONCLUSIONS: Most surveillance PPD testing of EM residents is performed at intervals recommended by the CDC. TB control programs at institutions sponsoring EM residencies need to improve both compliance with PFR mask fit testing by EM residents and availability of approved PFR masks in appropriate areas of the ED. Despite poor compliance with personal respiratory protection in ED patient encounters at risk for TB transmission, the risk of an EM resident's developing active TB over a three-year residency is low.

Growth of astrocytomas in the human tumor clonogenic assay and sensitivity to mismatched dsRNA and interferons.

Nine astrocytoma specimens were received from seven patients and processed for testing in the human tumor clonogenic assay (HTCA). Cells derived from these specimens were challenged with human natural alpha-interferon (alpha-IFN) and beta interferon (beta-IFN), recombinant beta interferon (beta ser-IFN), and mismatched double-stranded (ds) RNA (Ampligen). Six of the astrocytoma specimens formed adequate colonies for drug sensitivity testing (greater than or equal to 30 colonies/plate), and all were high-grade (III-IV) tumors. Sensitivity was defined as a greater than or equal to 50% decrease in tumor colony formation following drug exposure and was observed with alpha-IFN (2/4), beta-IFN (3/4), and mismatched dsRNA (4/5) exposure. No decrease in colony growth was observed after recombinant beta ser-IFN exposure, and in 2 of 3 cases, colony formation was stimulated. The sensitivity of 75 non-CNS solid tumors to mismatched dsRNA was compared to the high-grade astrocytomas in the HTCA. Of the 10 additional histologic tumor types studied, carcinoid and renal cell carcinomas exhibited the greatest sensitivity to mismatched dsRNA: 63% and 52%, respectively. However, in comparison, 80% of the high-grade astrocytomas were sensitive, demonstrating that these gliomas are among the most sensitive of human tumors to mismatched dsRNA in vitro. Clinical trials of interferons and mismatched dsRNA, coupled with in vitro sensitivity studies, should further define their therapeutic potential.

Synergistic inhibition of AZT-resistant HIV by AZT combined with poly(I):poly(C12U), without synergistic toxicity to bone marrow progenitor cell elements.

Mutation of human immunodeficiency virus (HIV) to drug resistance is an obstacle to HIV containment, and may account for the transitory nature of the improvement in CD4 cell counts of patients receiving azidothymidine (AZT). The emergence of AZT-resistant (AZTR) virus might be suppressed if a second therapeutic could be added; however, such a regimen would have to confer not only additional control over HIV replication but also no additional toxicity, especially to bone marrow progenitor cells. In the present study, HIV was isolated from patients receiving AZT alone and was studied for sensitivity to the mismatched double-stranded RNA, poly(I):poly(C12U) (ampligen). In addition, the combination of poly(I):poly(C12U) plus AZT was studied in vitro for toxicity to bone marrow CFU-GM and in patients receiving combined therapy for bone marrow toxicity. HIV isolated from patients receiving AZT alone showed higher resistance to AZT than wildtype virus, but remained sensitive to poly(I):poly(C12U). Poly(I):poly(C12U) and AZT were synergistic in inhibiting all isolates of HIV tested, regardless of their AZTR phenotype. Furthermore, the combination of poly(I):poly(C12U) and AZT showed no toxicity in vitro to bone marrow CFU-GM compared to AZT alone. In 11 HIV infected individuals receiving the combinational regimen, bone marrow function gradually improved. These results indicate that poly(I):poly(C12U) was active against AZTR HIV, synergistic with AZT and did not convey added toxicity.

An hypothesis concerning optimal therapy in HIV disease.

Ampligen, a mismatched double stranded RNA, is hypothesized to be an ideal base therapy for HIV disease to which other agents, such as the nucleoside analogue, AZT, can be advantageously added. The unique properties of Ampligen which support this hypothesis include activation of immune cells, inhibition of virus replication by inducing an antiviral cellular state and inhibition of growth of neoplastic cells. Ampligen is synergistic with other agents being used or being tested for use in HIV disease and is without toxicity.