Naturally occurring lymphocyte-mediated immunity to endogenous type-C virus in the mouse. Blocking of the lymphocyte reactivity with antisera to the virus.

The natural immune response in mice to their endogenous type-C viruses involves a complex interaction between cellular and humoral immune mechanisms. The virus-specific immune reactivities are a function of age and appear only subsequent to endogenous virus expression. Cellular immune activity was found to reside in a population of lymphocytes that were characterized as natural killer cells based on their absence of theta surface antigens or immunoglobulin or complement receptors. Cellular and humoral virus-specific immune responses co-occur in the same animal and pretreatment of virus-positive target cells with sera from virus-positive aging mice is capable of partially blocking the cytotoxic activity of reactive lymphocytes. The blocking activity of sera from individual mice increases as a function of age and endogenous virus expression and is highly correlated with the virus-specific complement-dependent cytotoxic activity of these sera. Mouse sera, whether naturally immune or immune as a result of hyperimmunization with type-C virus, exhibit blocking activity that can be removed by absorption with purified type-C virus or purified viral glycoprotein (gp 70) but not by absorption with noninfected syngeneic cells. High-titered and highly specific antisera directed against certain individual R-MuLV structural proteins reveal blocking activity. Monospecific antisera to gp 70 and p 12 exhibited high-titered blocking reactivities which are absorbable by the respective purified proteins. Blocking activity of antisera directed against other viral structural proteins could not be excluded with certainty. These findings raise the possibility that immunity in the mouse to endogenous type-C virus or virus-infected cells involves competition between serum-blocking activity and natural-killer cell activity and further provides a unique model system for studying the mechanism of action of blocking antisera known to have monospecific reactivity against defined and purifiable transplantation antigens.

Immunochemotherapy of transplantable Moloney leukemia with cyclophosphamide and allogeneic spleen lymphocytes and reversal of graft-versus-host disease with alloantiserum.

Histoincompatible (H-2) spleen cells from C57BL/6 mice that were sensitized with Moloney sarcoma virus caused fatal graft-versus-host disease in BALB/c mice when the cells were used in conjunction with an immunosuppressive, chemotherapeutic dose of cyclophosphamide to treat transplantable Moloney virus-induced leukemia. When antiserum against the donor spleen cells was administered 2 days after immunochemotherapy, graft-versus-host disease was prevented, but no immunotherapeutic effect was observed. When the antiserum was delayed for 3 days or more, lethal graft-versus-host disease occurred. Substitution of Moloney sarcoma virus-sensitized BALB/c X C57BL/6 F1 (hereafter called CB6F1) spleen cells for C57BL/6 cells, in conjunction with cyclophosphamide, "cured" 70% of the treated mice (accumulated value of two experiments). When anti-CB6F1 serum (alloantiserum) was administered 2 days after immunochemotherapy, the immunotherapeutic effect was abolished. Alloantiserum was not able to reverse the immunotherapeutic effect 3 days postgrafting or later, and there resulted a high percentage of long-term survivors. About two-thirds of the cured mice had positive donor-specific gamma-globulin titer 8 weeks postgrafting.

Drug-mediated immunogenic changes of virus-induced leukemia in vivo.

LSTRA and RBL-5 lymphomas induced by Moloney and Rauscher leukemia viruses, respectively, were used to determine whether antigenically altered tumors induced by 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide in vivo would retain their original antigenic properties and/or have new antigenic properties. The tumors became highly immunogenic in the syngeneic hosts after 4 to 8 transplant generations with drug treatment. Syngeneic mice could be protected against challenge with the parental tumor by presensitization with the drug-altered sublines while unrelated tumor lines were incapable of protecting them. The drug-altered subline of LSTRA was used for treatment of the LSTRA in conjunction with chemotherapy, and this immunochemotherapy produced significant increases in number of survivors and increases in median survival time compared to either treatment alone. Tolerance studies indicated that there are novel antigens and parental tumor antigens associated with the drug-treated sublines.

Efficacy of Coxiella burnetii and its chloroform-methanol residue (CMR) fraction against Rift Valley fever virus infection in mice.

Strains of Coxiella burnetii phase I and II whole cells (WC-I and WC-II) or whole cell fractions were assessed for their potential to induce long-lasting protection in endotoxin-non-responder C3H/HeJ or CD-1 mice against Rift Valley fever (RVF) virus challenge. Among the whole cell fractions, only the chloroform-methanol residue (CMR), administered as a single dose (100 micrograms per mouse) 24 h before viral challenge, effectively protected 100% of the mice from RVF virus; the CMR of the Ohio strain of C. burnetii was not protective. Most of the RVF virus-infected mice treated with other C. burnetii cell fractions died, although their times to death varied. Lipopolysaccharide (LPS) associated with CMR preparations used in these studies, did not protect against RVF virus challenge. A single dose of 100 micrograms of CMR given 24 h before viral challenge completely eradicated 4-5 logs of RVF virus in the serum, liver, spleen, and central nervous system. Compared to several other immunomodulators, CMR was an equally effective antiviral agent. Efficacy of CMR of both Henzerling and Ohio strains disappeared or was marginal when treatment was initiated 2-3 days before RVF viral challenge, even when a second or a third dose of CMR was administered after challenge. A single dose of liposome-encapsulated CMR to RVF virus-infected mice extended the range of therapeutic efficacy of this biologically active component of C. burnetii to 4 days before infection.

Antiviral and immunomodulating inhibitors of experimentally-induced Punta Toro virus infections.

A major component of a US Army Medical Research and Development Command-supported program to discover and develop new drugs for the treatment of Rift Valley fever, sandfly fever, and Crimean-Congo hemorrhagic fever has been to study candidate test materials against hepatotropic infections of C57BL/6 mice induced by the related but less biohazardous Punta Toro virus (PTV). The effects of 75 compounds, some of which were considered immunomodulators in their primary mechanism of activity, were studied in the PTV infection model. Of these, ribavirin, ribamidine, ribavirin 2',3',5'-triacetate, tiazofurin, tiazofurin-5'-monophosphate, tiazofurin-2',3',5'-triacetate, selenazofurin, pyrazofurin, 3-deazaguanine, and 3-deazaguanosine were considered significantly inhibitory, acting against the infection by a direct antiviral (non-immunomodulatory) fashion. These compounds had therapeutic indices (TI) ranging from > or = 5 to 65, using increased survivors as the evaluation parameter. Immunomodulators considered significantly inhibitory to this infection were poly (ICLC), ampligen, human recombinant interferon-alpha-A/D, MVE-1, MVE-2, AM-3, AM-5, mannozym, bropirimine, CL246,738, phenyleneamine, and 7-thia-8-oxoguanosine. Utilizing increased survivor numbers as measure of activity, these inhibitors had TI ranging from > or = 16 to 1000. Other antiviral effects exerted by the active compounds included reduction of hepatic icterus, lowered serum glutamic oxaloacetic and pyruvic acid transaminases, and inhibition of recoverable serum and liver virus titers. The active immunomodulators were significantly effective when therapy was initiated as late as 48 h after virus inoculation, at a time when clinical signs of the PTV disease were being manifested in the animal.

Effect of human, recombinant interleukin 2 on Punta Toro virus infections in C57BL/6 mice.

The effect of human recombinant interleukin-2 (rIL-2) on Punta Toro virus (PTV) infection was investigated in C57BL/6 mice. Immunologic and viral parameters were assessed after mice were treated i.p. with rIL-2 for 5 days. Treatment of mice with 25000 and 12500 units/mouse of rIL-2 resulted in significant inhibition of the disease as indicated by increases in survival of mice as well as decreases in liver and serum virus titers. Serum glutamic oxalic acid and pyruvic acid transaminase levels were also lowered indicating reduced liver damage. Murine IL-2 production returned to normal or above-normal levels in rIL-2 treated mice. Natural killer cell activity was also moderately stimulated by rIL-2 treatment. Significant amounts of interferon were not detected in the sera of treated mice. Weight gain and survival rates were similar for both toxicity and normal controls indicating that rIL-2 treatments had no toxic effect.

Antiviral immunotoxins: antibody-mediated delivery of gelonin inhibits Pichinde virus replication in vitro.

Immunotoxins were produced and evaluated for antiviral activity against Pichinde virus, a member of the family Arenaviridae. Immunoglobulins were conjugated to the ribosome-inactivating protein, gelonin, through a disulfide linkage to form the immunotoxins. Immunotoxins were produced utilizing monoclonal antibodies, immunoglobulin-binding proteins and hyperimmune sera. An immunotoxin consisting of hyperimmune rabbit sera conjugated with gelonin displayed strong antiviral activity against Pichinde virus, as did a protein G-gelonin indirect immunotoxin in combination with nonconjugated hyperimmune sera. Hyperimmune rabbit sera conjugated with gelonin caused no detectable cytotoxicity in non-infected Vero cells as measured by [3H]leucine incorporation. The 50% effective dose for the immunotoxin was 0.018 microM compared with 86 microM for ribavirin.

Characterization of the non-specific humoral and cellular antiviral immunity stimulated by the chloroform-methanol residue (CMR) fraction of Coxiella burnetii.

Modulation of the immune response by the chloroform-methanol residue (CMR) of phase I Coxiella burnetii whole cell was studied in Rift Valley fever virus-infected, or in naive endotoxin-non-responder C3H/HeJ mice. A single dose of CMR completely protected the mice from viral infection. Treating virus-infected mice with antibodies directed against interferons alpha/beta (IFN-alpha beta) and gamma (IFN-gamma) eliminated the CMR-induced protection. CMR stimulated the production of high levels of IFN-alpha/beta and 2'-5'-oligoadenylate synthetase activities in sera of the CMR-treated mice. IFN-gamma was present in supernatants of cultured spleen cells of CMR-treated, virus-infected mice, but not in their serum. Priming mice with CMR optimized the release of INF-gamma, interleukin-1 alpha (IL-1 alpha) and IL-6 from splenocytes in vitro. When stimulated in vitro, IL-2 and granulocyte-macrophage stimulating factor (GM-CSF) did not require in vivo priming for release from cultured spleen cells. Fluorescence-assisted cytometry of CMR-treated mouse spleen cells showed there was a CMR-dependent increase in the percentage of T-cells and Ia-positive T-cells. There also was a biphasic increase in the ratio between Th (L3T4) and Ts (Lyt2) cells. Biological activities stimulated by CMR indicate that CMR is a potent immunostimulant, which may modulate specific and non-specific antiviral responses.

A newly developed immunofluorescent assay for determining the Pichinde virus-inhibitory effects of selected nucleoside analogues.

An immunofluorescent assay (IFA) for Pichinde virus (PCV), a member of the family Arenaviridae, was developed for antiviral drug assays against the virus. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to virus-infected cells at 24 h after the initial infection and counting the infected cells with an epifluorescence microscope. The average 50% effective dose (ED50) for a series of nucleoside analogues tested against PCV using this IFA was: 2-beta-D-ribofuranosylselenazole-4-carboxamide (selenazofurin), less than 1.0 microgram/ml; 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 6.0 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide- 5'-phosphate hydrate (ribavirin-5'-monophosphate), 15.8 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5'-hemisuccinate (ribavirin-5'-hemisuccinate), 14.7 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5'-(2,3- dimethyl)hemisuccinate [ribavirin-5'-(2,3-dimethyl)hemisuccinate], 213.5 micrograms/ml; 4-hydroxy-1-beta-D-ribofuranosyl-2-pyridone (3-deazauridine), 5.2 micrograms/ml; and (S)-9-(2,3-dihydroxypropyl)adenine, ([S]-DHPA), 471.0 micrograms/ml. In comparison, the ED50 of ribavirin using inhibition of marginal PCV-induced cytopathogenic effect after 12 days was 6.0 micrograms/ml and using plaque reduction after 5 days was 2.5 micrograms/ml, indicating that this IFA was of comparable sensitivity to these other tests.

Chemotherapeutic prevention of complications caused by vaccinia virus-vectored immunogen.

Vaccinia virus strains and constructs differ greatly in the number of PFUs required to produce tail lesions in the vaccinia virus mouse model. The pathogenesis of lesion formation appeared to involve virus spread from an initial focus in specific cells surrounding hair follicles to other concentrated areas of the dermis and finally, at the time of lesion development, to the epidermis. Antivirals that suppressed tail lesions, to a greater or lesser degree, included ara A, ribavirin, rifampicin, adenosine N'-oxide, and selected analogues. Immunomodulators, including ampligen and recombinant interferon, suppressed lesions at very low doses. Spread of virus infection from the dermis to the epidermis was inhibited as determined by immunofluorescence. These studies in the tail lesion model have suggested drugs that could be tested further in primate models of vaccinia virus infection. In addition, these studies provide additional data on a model that may be a useful adjunct in safety testing of recombinant vaccinia virus vaccines.

Injection of inactivated phase I Coxiella burnetii increases non-specific resistance to infection and stimulates lymphokine production in mice.

The encounter of phase I C. burnetii with the host results in seemingly disparate consequences. On the one hand, in vitro lymphocyte responses to mitogens and homologous recall antigen are suppressed. On the other, host resistance to a variety of infectious agents and to a tumor is increased. An explanation for this augmented immune response surely involves the ability of C. burnetii to stimulate cytokines, such as interferon and TNF, which enhance host immune function.

Potential role of immunomodulators for treatment of phlebovirus infections of animals.

Rift Valley fever (RVFV) is a major phlebovirus-induced epizootic disease of domestic animals (primarily cattle and sheep) in Africa. No therapies for the disease are known. A related phlebovirus, Punta Toro virus (PTV), has been adapted to induce an RVFV-like disease in C57BL/6 mice. This PTV infection has been used as a model for RVFV because it is reasonably safe and does not require high-level biologic containment. The infection model has been used to study the potential role of immunomodulating substances as therapies. A spectrum of immunomodulators has been studied; those immunomodulators most capable of preventing death and other disease manifestations are ampligen, bropirimine, poly (ICLC), AM-3, P-136, and 7-thia-8-oxoguanosine. An immunologic parameter common to all these substances has been their ability to induce interferon. Timing studies have indicated that these active substances may be administered therapeutically as well as prophylactically to inhibit markedly the progress of the disease. Further work is needed in the development of these materials for use in treating viral infections in domestic animals. As a next step, studies need to be run to compare the immunologic profiles induced by each substance in domestic animals and in mice.

Long-term treatment of malignant gliomas with intramuscularly administered polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose: an open pilot study.

Polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC) (10-50 mcg/kg, administered intramuscularly one to three times weekly) was given for < or = 56 months to 38 patients with malignant gliomas. There was minimal or no toxicity. Twenty of 30 patients (66%) receiving at least twice weekly poly-ICLC showed regression or stabilization of gadolinium-enhancing tumor, as revealed by magnetic resonance imaging (median = 65% volume decrease). All but one patient with anaplastic astrocytomas who received continuous poly-ICLC remain alive, with a median progression-free survival of 54 months from diagnosis. Median Kaplan-Meier survival is 19 months for patients with glioblastomas who receive at least twice weekly poly-ICLC treatments. Tumor response was associated with 2',5' -oligoadenylate synthetase activation (P = 0.03) but not with serum interferon. We hypothesize clinical activation by poly-ICLC of a basic host tumor suppressor system. Prolonged, quality survival with tumor stabilization or regression confirmed by magnetic resonance imaging for most patients with anaplastic astrocytomas and glioblastomas suggests that more extensive laboratory and controlled clinical studies are warranted. The concept of long-term, broad spectrum stimulation of host defenses with nontoxic, inexpensive double-stranded ribonucleic acids, such as low-dose poly-ICLC, may be applicable to the treatment of other malignancies.

Role of macrophages in the expression of immune responses.

Cells of the mononuclear phagocyte system have a crucial role as affector and effector cells in the body's defense against foreign cells and microorganisms. Macrophages function as the first line of defense via phagocytosis or opsonic phagocytosis as early as the promonocytic stage of their development. Macrophages act as affector cells via antigen presentation to lymphocytes, and they participate in the activation of T and B lymphocytes through the secretion of lymphostimulatory substances (monokines). In the cycle of reciprocal interactions, macrophages are themselves being activated via the secretory products of the lymphocytes--the lymphokines. Activated macrophages are endowed with effector functions exerted by their tumoricidal, microbicidal, and suppressor activities. Undoubtedly, additional research will enhance the importance and application of this unique cell type with multiple functions.

Different electrophoretypes of human rotaviruses in Hungary.

Polyacrylamide gel electrophoresis of rotaviral double-stranded ribonucleic acid (RNA) extracted directly from faecal specimens collected in three different parts of Hungary was applied to characterize and distinguish 21 randomly selected viral isolates. This technique made it possible to define 7 different electrophoretypes. Of the isolates 11 exhibited an identical "long" electrophoretic migration pattern. "Short" RNA pattern was found in two cases, and one atypical rotavirus was also revealed. This is the first description of rotavirus RNA electrophoretypes in Hungary.

Superiority of traditional village diet and lifestyle in minimizing cardiovascular disease risk in Papua New Guineans.

In the traditional society of Papua New Guinea (PNG) atherosclerotic cardiovascular diseases (CVD) are rare. However, among the urban population reports of cases of atheroma-related CVD are increasing. The purpose of this study was therefore to compare the CVD risk factors in a homogeneous population of the Southern Highlands Province living in both rural and urban areas differing only in their diet and lifestyle. A total of 221 Samberigi people over the age of 25 years were selected for the survey. These included 123 individuals from remote villages of Samberigi and 98 of their relatives who had lived in Port Moresby city continuously for a minimum of 5 years. The anthropometric measurements, blood lipid, blood glucose and glycosylated haemoglobin (HbA1c) levels were measured and compared. The rural diets were mainly of vegetarian type, limited in variety and low in fat and protein content. In the urban subjects, the typical meal comprised refined foods with high fat and protein content. The urban men and women had significantly (p < 0.05) greater body weight, body mass index (BMI), and waist and hip circumferences than their rural counterparts. In Port Moresby, 57% of the men and 67% of the women were overweight or obese compared to 28% of their rural counterparts. Similarly, the mean plasma total cholesterol, low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDLC), fasting blood glucose and HbA1c were significantly higher in the urban group. However, no significant differences were demonstrated for waist to hip ratio, LDLC/HDLC ratio and lipoprotein (a) levels between the two groups. The total cholesterol, LDLC and HbA1c were positively associated with age and BMI in both rural and urban locations. In conclusion, there were significant increases in CVD risk factors in the urban population compared to the rural residents. This was predominantly due to the adoption of a western lifestyle and diet as people moved from rural villages to the city of Port Moresby.

Evaluation of thyroid stimulating hormone (TSH) alone as a first-line thyroid function test (TFT) in Papua New Guinea.

In the Port Moresby General Hospital, the Chemical Pathology Department assays both thyroid stimulating hormone (TSH) and free thyroxine (FT4) on all requests for a thyroid function test (TFT). The cost of assaying both tests is obviously higher than either test alone. In order to minimize the cost of a TFT we aimed to determine if TSH or FT4 alone as a first-line test would be adequate in assessing the thyroid hormone status of patients. We analyzed TFT records from January 1996 to May 2000 in the Port Moresby General Hospital. A total of 3089 TSH and 2867 FT4 were assayed at an annual reagent cost of Papua New Guinea kina 14,500. When TSH alone is used as a first-line test at the Port Moresby General Hospital, the biochemical status of 95% of patients will be appropriately categorized as euthyroidism, hypothyroidism or hyperthyroidism with only 5% discrepant (ie, normal TSH with abnormal FT4) results. In contrast, using FT4 alone as a first-line test correctly classifies only 84% of TFTs. Euthyroid status is observed in 50% of patients and FT4 assays on these samples will be excluded appropriately if a TSH-only protocol is adopted. Furthermore, we will save a quarter of the yearly cost of TFTs on reagents alone by performing TSH only. We conclude that TSH alone is an adequate first-line thyroid function test in Papua New Guinea and when it is normal no further FT4 test is necessary unless clinically indicated.

Review of cases of hyponatraemia in the Port Moresby General Hospital between August 1993 and June 1995.

Hyponatraemia (serum sodium level below 130 mmol/l) is a common electrolyte abnormality in a hospital population. It can be associated with dehydration, overhydration or normal hydration. Clinically, it is important to recognize the common diseases associated with hyponatraemia since correct treatment in terms of fluid replacement is essential in preventing complications of low serum sodium. We have reviewed results of serum sodium tested from patients admitted to the Port Moresby General Hospital between 1993 and 1995. This was aimed at identifying the most common features associated with low sodium. Clinical information and diagnosis were obtained by looking through a series of request forms. Of the approximately 30,000 blood samples taken over 23 months, the percentage of samples with hyponatraemia was about 1%. Hyponatraemia was more common in medical (38%) and paediatric (35%) cases and at the extremes of ages, ie, under the age of 6 years and above 40 years. Over a quarter of the hyponatraemic patients had severe hyponatraemia (serum sodium below 120 mmol/l). Clinical conditions commonly associated with hyponatraemia, in descending order of importance, were diarrhoea and vomiting, renal failure, central nervous system infections and trauma, pulmonary infections, oedematous states (eg, nephrotic syndrome) and diabetes mellitus.