Adenovirus tumor-specific transplantation antigen is a function of the E1A early region.

Viable recombinant adenoviruses that carry a portion of the type 12 E1A and E1B transcription units in a type 5 background were used to identify genes controlling expression of the adenovirus tumor-specific transplantation antigen (TSTA). The TSTA immunity is not crossreacting between the group A and group C adenovirus serotypes. Viruses carrying the E1A region (sub370-12E1A), or both E1A and E1B (sub370-12E1AB) regions of Ad12, induce a strong transplantation immunity against tumors induced by syngeneic cells transformed with adenovirus 12, but fail to induce any protection against syngeneic cells transformed with adenovirus 2. Immunization with the virus carrying only the E1B region (sub370-12E1B) of adenovirus 12 induces no immunity to adenovirus 12 transformed cell line, but confers a strong protection against cells transformed with adenovirus 2. These results provide strong evidence that the adenovirus tumor-specific transplantation antigen is a function of the E1A early region.

Requirement for the adenovirus type 9 E4 region in production of mammary tumors.

Oncogenic viruses demonstrating a strict tropism for the mammary gland provide special opportunities to study the susceptibility of this tissue to neoplasia. In rats, human adenovirus type 9 (Ad9) elicits mammary fibroadenomas that are similar to common breast tumors in women, as well as phyllodes-like tumors and mammary sarcomas. By constructing recombinant adenoviruses between Ad9 and Ad26 (a related nontumorigenic virus), it was shown that the Ad9 E4 region was absolutely required to produce these mammary tumors. This indicates that an adenovirus gene located outside the classic transforming region (E1) can significantly influence the in vivo oncogenicity of an adenovirus. Consistent with a direct role in mammary gland oncogenesis, the Ad9 E4 region also exhibited transforming properties in vitro. Therefore, the Ad9 E4 region is a viral oncogene specifically involved in mammary gland tumorigenesis.

Increased levels of glycoproteins containing mannose 6-phosphate in human breast carcinomas.

Newly synthesized enzymes destined for lysosomal localization contain mannose 6-phosphate (Man6-P) residues, allowing interaction with Man6-P receptors (MPRs) and subsequent intracellular targeting to the lysosome. In most cultured cells, lysosomal enzymes are rapidly dephosphorylated after targeting, but in some transformed cell lines, these proteins retain the Man6-P marker. To investigate the significance of this in human malignancy, we examined the persistence of the Man6-P marker in human breast biopsy specimens using MPR derivatives as affinity probes. In one approach, extracts of frozen tissue were standardized to protein content, fractionated by SDS-PAGE, immobilized on nitrocellulose, and probed with iodinated MPR. On average, carcinomas contained 4-fold higher levels of Man6-P glycoproteins than did benign tumors or normal breast samples. In about 15% of the carcinomas, levels of Man6-P glycoproteins were highly elevated (7-10-fold). Multiple Man6-P glycoproteins were detected, suggesting a general alteration in the synthesis or processing of many lysosomal enzymes in carcinomas. In a second approach, sections of formalin-fixed breast biopsy specimens were probed with biotinylated MPR. Malignant cells in 25 of 75 carcinomas exhibited granular cytoplasmic staining in what appears to be intracellular vesicles. Staining was specifically inhibited by Man6-P and was not observed in stromal components or lymphocytes. In addition, Man6-phosphorylated proteins were not detected in the 14 normal or benign biopsy samples examined. Staining appeared to be independent of most prognostic factors examined, including p53, cathepsin D, DNA ploidy, and hormone (estrogen and progesterone) receptor status. However, positive staining was significantly associated with high histological and nuclear grades (P < 0.05) and potentially with c-erbB-2 (P < 0.10), suggesting that elevated levels of Man6-P glycoproteins are associated with the more aggressive tumors.

Alpha-interferon structure and natural killer cell stimulatory activity.

Expression vectors for human alpha-interferon (Hu-IFN-alpha) J1, a site-specific mutant [Ser116]Hu-IFN-alpha J1, and Hu-IFN-alpha J/C or Hu-IFN-alpha C/J hybrids were constructed and expressed in Escherichia coli. These interferons and others were purified by immunoaffinity chromatography with a monoclonal antibody against human alpha-interferon. Their antiviral activity and ability to stimulate natural killer cell activity were determined in comparison to several other human interferons. These results provide some insight into structure-activity relationships for stimulating natural killer cells and confirm our previous conclusions that antiviral activity cannot be used to predict other activities for an individual IFN-alpha species. The observations suggest that the tertiary structure rather than any specific linear sequence of amino acids regulates the ability of the interferons to stimulate natural killer cell activity.

Immune thrombocytopenia in hemophiliacs infected with human immunodeficiency virus and their response to splenectomy.

We studied five patients with hemophilia A in the age range of 18 to 64 years who were infected with human immunodeficiency virus and who developed immune thrombocytopenia. The clinical course of immune thrombocytopenia in relation to human immunodeficiency virus infection and the patients' responses to splenectomy and immune variables were determined. All five patients developed antibody to human immunodeficiency virus 6 to 60 months (median, 24 months) before the onset of thrombocytopenia, and two patients became human immunodeficiency virus antigenemic (one patient at the onset of immune thrombocytopenia and the other 60 months after the onset of immune thrombocytopenia [24 months after splenectomy]). All five patients had a strong platelet-associated immunoglobulin G and three patients also had a weak platelet-associated immunoglobulin M on their platelets. In four of five patients danazol therapy failed, and three patients required moderate doses of prednisone. Because of the progression of immune thrombocytopenia, four of the five patients underwent splenectomy with preoperative high-dose intravenous immune globulin. All four had an excellent immediate response to splenectomy, with a rise in platelet count to more than 300 x 10(9)/L and sustained remission during postsplenectomy follow-up of 6 to 45 months. There was no significant drop in CD4 and CD8 counts after splenectomy, and all four patients remained clinically well.

B-cell activation and immunoregulation in end-stage renal disease patients receiving hemodialysis.

B-lymphocyte functions were studied in peripheral blood mononuclear cells of end-stage renal disease patients undergoing intermittent hemodialysis for longer than two years. T-cell-dependent B lymphocyte proliferation after pokeweed mitogen stimulation was low in half of the hemodialyzed patients. T cell-independent B cell response to Staphylococcus aureus, Cowan I, was also significantly reduced. Spontaneous production of immunoglobulin in cultures of peripheral blood mononuclear cells of uremic patients was comparable with that of healthy controls, but pokeweed mitogen-stimulated antibody secretion was significantly reduced with cells from patients undergoing hemodialysis. Helper T-cell functions in B-cell activation were also qualitatively deficient in uremic patients. It is concluded that B-cell activation and immunoregulation is defective in patients undergoing long-term hemodialysis.

Chimerasome-mediated gene transfer in vitro and in vivo.

Proteoliposome delivery vesicles can be prepared by the protein-cochleate method [Gould-Fogerite and Mannino, Anal. Biochem. 148 (1985) 15-25; Mannino and Gould-Fogerite, Biotechniques 6 (1988) 682-690]. Proteins which mediate the entry of enveloped viruses into cells are integrated in the lipid bilayer, and materials are encapsulated at high efficiency within the aqueous interior of these vesicles. We describe proteoliposome-mediated delivery of proteins and drugs into entire populations of cells in culture. Material can be delivered gradually by Sendai-virus-glycoprotein-containing proteoliposomes. Alternatively, synchronous delivery to a population can be achieved by exposing cell-bound influenza glycoprotein vesicles briefly to low pH buffer. When DNA is encapsulated, chimeric proteoliposome gene-transfer vesicles (chimerasomes), which mediate high-efficiency gene transfer in vitro and in vivo, are produced. Stable expression of a bovine papilloma virus-based plasmid in tissue-cultured cells, at 100,000 times greater efficiency than Ca.phosphate precipitation of DNA, with respect to the quantity of DNA used, has been achieved. Stable gene transfer and expression in mice has been obtained by subcutaneous injection of chimerasomes containing a plasmid expressing the early region of polyoma virus. In one experimental group, 50% of the mice developed tumors which were shown to express polyoma virus early proteins and contain the transferred DNA. This is the first report of stable gene transfer in animals mediated by a liposome- or proteoliposome-based system.

Human immunodeficiency virus infection in sexually active wives of infected hemophilic men.

PURPOSE: Because of past multiple exposures to contaminated coagulation factor concentrates, the prevalence of human immunodeficiency virus (HIV) infection among adult hemophilic men in the United States is reported to range from 75 to 90 percent. The risk of HIV transmission through a long-term monogamous heterosexual contact can be estimated by studying the spouses of hemophilic subjects since these couples generally do not abuse intravenous drugs, usually maintain stable monogamous relationships, and are usually free of other risk factors. Our purpose was to gather data on the risk of heterosexual transmission of HIV infection in the context of long-term monogamous relations according to the duration of HIV antibody seropositivity and of HIV antigenemia in HIV-infected hemophilic men, and their sexual habits. SUBJECTS AND METHODS: Infection with HIV was studied in 14 sexually active spouses of infected hemophilic men who had been HIV antibody reactive for a mean of 46 +/- 23 (SD) months. One half of the hemophilic men studied had overt HIV antigenemia for a mean duration of 27 +/- 23 (SD) months; six of the men studied fulfilled clinical criteria for the diagnosis of acquired immunodeficiency syndrome (AIDS). All 14 couples were sexually active in a strictly monogamous fashion, in marriages of 13.5 +/- 10.5 (SD) years with an average reported frequency of four sexual encounters per month (range: one to 12). Plasma samples of the hemophilic husbands were retrospectively analyzed for HIV and hepatitis B virus markers. Blood samples were obtained from female spouses on at least two occasions, six months apart. Comprehensive questionnaires regarding sexual habits and other risk factors were filled out by each couple; during this interview, the couple was counseled about safe sexual practices. None of the couples studied used condoms prior to January 1986. RESULTS: Antibodies to HIV developed in only one of the 14 wives. At the time when this seroconversion was detected, her husband, in whom AIDS developed, had been reactive for HIV antibody for 49 months, and showed positive findings for HIV antigen for 26 months. No additional risk factors were identified for this couple. The infected female spouse, however, has a 14-year history of multiple sclerosis, and had been treated with immunosuppressant intermittently. Despite a significantly reduced number of CD4 lymphocytes, she has remained clinically asymptomatic for 17 months since seroconversion. HIV antibodies did not develop in any of the 13 remaining wives, despite the frequent practice of oral sex by six couples and reports of occasional anal intercourse by another couple.(ABSTRACT TRUNCATED AT 400 WORDS)

Acquired immune deficiency syndrome. No evidence of the presence of cyclosporine.

It has been postulated that the acquired immune deficiency syndrome (AIDS) may be a result of the systemic presence of an immunosuppressive cyclosporine-like molecule released in chronic fungal infections. This possibility was examined by analysis of blood, plasma, or serum samples obtained from AIDS patients, from subjects with prodromal AIDS, and from healthy subjects belonging to several of the recognized "AIDS risk groups" for cyclosporine-like substances. The sensitivity of the analytic methods and the stability of cyclosporine during the storage of blood were verified by analysis of blood specimens obtained from a normal volunteer after oral ingestion of various doses of cyclosporine. Radioimmunoassay, high-pressure liquid chromatography, and analysis by combination of these two methods failed to detect cyclosporine or cyclosporine-like substances in subjects with established or prodromal AIDS and in AIDS-free persons belonging to the risk groups. These results indicate that the breakdown of cellular immunity in AIDS is not due to circulating cyclosporine.

Suppressor cells in end-stage renal disease. Functional assays and monoclonal antibody analysis.

Suppressor cell activity after concanavalin A induction was studied in peripheral blood mononuclear cells of patients undergoing long-term hemodialysis. Suppression both of the mixed lymphocyte reaction and of allogeneic cells stimulated with phytohemagglutinin was significantly higher with peripheral blood mononuclear cells from patients undergoing hemodialysis than with cells from control subjects. Expression of the Ia antigen on T lymphocytes (associated with immunologic activation) was studied by staining with monoclonal antibodies and two-color fluorescence analysis in a computer-linked cytofluorograph. In unstimulated cells, there was no significant difference between the patients and control subjects. After concanavalin A induction, the percentage of T4, and particularly of T8, cells expressing the Ia antigen was significantly higher in the group undergoing hemodialysis. The functional suppression seen after concanavalin A induction in the mixed lymphocyte reaction was significantly reduced by treatment with OKT8 monoclonal antibody and complement; in phytohemagglutinin cultures, both OKT8 and OKIa*1 antibodies were effective. The reduced in vitro response of uremic lymphocytes may thus be a consequence of increased suppressor activity associated with the T8-positive, Ia-positive subset of T cells.

T cell subsets and cellular immunity in end-stage renal disease.

The T lymphocyte population was studied by immunofluorescent staining with monoclonal antibodies and laser flow cytometry in the blood of 50 patients with end-stage renal disease undergoing long-term maintenance intermittent hemodialysis. The absolute number of T cells was lower in patients receiving dialysis for more than one year (p less than 0.001), as was the absolute count of helper T cells (p less than 0.005). In patients under 30 years of age, the absolute number of helper T cells was markedly reduced, whereas the number of suppressor/cytotoxic T lymphocytes was not changed. In patients between the ages of 30 and 60 years, both helper and suppressor cells were significantly reduced. In patients over 60 years of age, only the number of helper T cells was reduced. The in vitro response of patients' lymphocytes was reduced both in the mixed lymphocyte reaction (p less than 0.01) and after phytohemagglutinin stimulation (p less than 0.001). Natural killer cytotoxicity of patients' peripheral blood mononuclear cells, however, was unaffected.

Responsiveness of preactivated B cells to IL-2 and IL-6. Effect of cyclosporine and rapamycin.

In order to determine which drug may be more effective in clinical abnormalities associated with polyclonal B lymphocyte activation, we compared the in vitro effects of CsA and rapamycin on proliferation or differentiation of preactivated B cells. For that purpose, highly purified B lymphocytes were preactivated in the presence of formalinized Staphylococcus aureus bacteria and then recultured in the presence or in the absence of either rIL-2, rIL-6, or combination or rIL-2 and rIL-6. After 48 hr in culture, S. aureus bacteria upregulated significantly the binding of phycoerythrin-conjugated IL-2 and IL-6, respectively, by purified B lymphocytes, indicating generation and/or upregulation of receptors for these cytokines. Such preactivated B lymphocytes proliferated in response to optimal concentrations of rIL-2, whereas the addition of rIL-6 to preactivated cells was always accompanied by a decrease of the proliferation rate. CsA upregulated cell proliferation when it was added in the second culture period in the presence or in the absence of rIL-6, whereas rapamycin had no effect in these cases. A combination of rIL-2 plus rIL-6 upregulated significantly the proliferative responses of preactivated B cells. In such cultures both CsA and rapamycin had an inhibitory effect on the proliferative responses. IgM production was unaffected by the addition of rIL-6 to cultures of preactivated B cells, whereas addition of rIL-2 and of the IL-2/IL-6 combination enhanced considerably IgM production. Irrespective of cytokines added, CsA upregulated the production of IgM. In contrast, rapamycin inhibited IgM production in all cases. Our results indicate that, in this experimental system, rapamycin is an effective immunosuppressive agent and its use, at least in vitro, is not accompanied by an upregulation of either the proliferation or differentiation of B lymphocytes.

Binding of phycoerythrin-conjugated interleukin-6 to in vitro-activated human peripheral blood mononuclear cells--effect of immunosuppressive agents and of a calcium channel blocker.

We studied the effect of cyclosporine A, prednisolone, and the Ca2+ channel blocker verapamil on interleukin-6 binding to mitogen-activated peripheral blood mononuclear cells, using a flow cytometric technique and phycoerythrin-conjugated IL-6. All mitogenic stimuli up-regulated IL-6 binding to a variable degree. PHA alone or in combination with PMA was the most effective stimulant in up-regulating IL-6 binding in all the experiments performed. The main changes in IL-6 binding were seen in the large cell cluster, which consisted mainly of lymphoblasts. PHA and PHA/PMA, however, also up-regulated the mean fluorescence intensity on the small cell cluster, which consisted mainly of quiescent lymphocytes. The overall effect of the three pharmacological agents on mitogen-up-regulated IL-6 binding was minimal; most significant were a down-regulation by all three agents of IL-6 binding by small lymphocytes in PHA/PMA cultures, a down-regulation of IL-6 binding by CsA in PHA/PMA-induced large PBMC, and an up-regulation by verapamil of PMA-induced IL-6 binding in large PBMC. Measurements of IL-2 binding and of IL-6 production in the same cultures showed a different pattern than that seen with IL-6 binding, as well as different CsA, prednisolone, and verapamil action. In conclusion, by using a new flow cytometric technique providing information both about the quantity of bound cytokine and about the proportion of IL-6-binding cells, we have demonstrated that IL-6 receptor expression in vitro by PBMC can be up-regulated by the use of stimulants differing in the signal transduction pathways they activate. In addition, by using different pharmacological agents and stimuli to dissect different activation pathways of the in vitro immune response, we conclude that IL-6R generation is regulated differently from IL-6 production. Furthermore, since CsA and prednisolone are known inhibitors of in vitro IL-2 production, our results indicate that IL-6R generation does not rely exclusively on the presence of IL-2.

Mixed lymphocyte reaction-induced release of soluble IL-2 receptor.

We studied the release of soluble interleukin 2 receptor (sIL-2R) by PBMC in mixed lymphocyte reaction (MLR) in order to clarify the significance of high plasma levels of sIL-2R in transplant patients undergoing rejection. Levels of sIL-2R were shown to increase progressively after the first day of the MLR and reached their peak on day 5. This pattern of sIL-2R correlated with the incorporation of [3H]thymidine. CsA and prednisolone (PRED) were added at the beginning of the MLR and were shown to inhibit the release of sIL-2R. This inhibition correlated with an inhibition of the [3H]thymidine incorporation. When CsA and PRED were added 24 hr after the initiation of the MLR, a similar inhibition of sIL-2R release was observed, but when they were added 48 hr after the initiation or in the last day of the MLR little or no effect was observed. Incubation of responder or stimulator-responder cells with either CsA or PRED before the initiation of MLR showed that only CsA preincubation was accompanied by decreased [3H]thymidine incorporation. Preincubation with CsA inhibited the release of sIL-2R, whereas PRED had a variable effect. Recombinant IL-2 was shown to augment the release of sIL-2R even at very low doses, but it did not alter significantly MLR-induced [3H]thymidine incorporation. The addition of rIL-2 at the initiation of the MLR was also shown to reverse completely the PRED inhibition of the MLR-induced release of sIL-2R and of the [3H]thymidine incorporation. Addition of rIL-2 reversed only partially CsA-induced inhibition. Addition of different concentrations of sIL-2R at the initiation of the MLR were not shown to affect incorporation of [3H]thymidine. We conclude that the release of sIL-2R in response to alloantigens is an IL-2-dependent phenomenon, and determination of its levels might be a useful indicator of either in vitro or in vivo alloantigen responses and of the effectiveness of immunosuppressive treatment.

Usefulness of transesophageal echocardiography in predicting mortality and morbidity in stroke patients without clinically known cardiac sources of embolus.

This study tested the hypothesis that stroke patients without a cardiac source of embolism suspected by clinical examination can be risk stratified by transesophageal echocardiography. Forty ischemic stroke patients without atrial fibrillation, prosthetic valves, ejection fraction < 20%, or recent myocardial infarction underwent multiplane transesophageal echocardiography: 24 (designated high risk) had > or = 1 of the following: left heart thrombus, vegetation, mass or spontaneous echo contrast, mobile ascending aortic or arch debris, patent foramen ovale, atrial septal defect or aneurysm, mitral annular calcification, mitral valve thickening, prolapse or mitral valve strands. End points were death, recurrent stroke, transient ischemic attack, myocardial infarction or peripheral embolism. Thirty-eight patients (95%) (23 high, 15 low risk) were followed for 14 +/- 8 months: 9 (24%) died of vascular causes including 4 who had a cardiac cause of death and 5 who had fatal strokes. Eight had recurrent strokes (4 nonfatal) and 1 nonfatal myocardial infarction occurred. Cardiovascular survival was predicted by transesophageal echocardiography: survival rates were 92% (low risk) and 63% (high risk) at 24 months (p = 0.036). Left atrial enlargement was independently associated with death from stroke (fatal stroke occurred in 25% of those with atrial enlargement compared to 8% of those with normal atrial dimension, p < or = 0.03), as was left atrial spontaneous echo contrast (50% died vs 9% without contrast, p < or = 0.03). Left ventricular hypertrophy and aortic atherosclerosis were both associated with the risk of recurrent stroke (30% of patients with ventricular hypertrophy had recurrent stroke compared to 10% with normal wall thickness (p < or = 0.05); 30% with aortic atherosclerosis had a recurrent stroke compared to none with a normal aorta (p < or = 0.05). Thus, transesophageal echocardiography clearly identifies patients at a high risk for cardiovascular mortality and morbidity after stroke despite an unsuspected source of embolism by clinical examination.

Diffuse large-cell lymphoma with monoclonal IgM kappa and cold agglutinin.

A patient with diffuse large-cell lymphoma associated with a serum monoclonal IgM kappa and a cold agglutinin is described. The cold agglutinin was the initial manifestation of disease and was apparent for at least two months before the diagnosis of lymphoma. The lymphoma cells had surface and cytoplasmic IgM kappa.

Clonal B-cell proliferation in an infant with congenital HIV infection and immune thrombocytopenia.

An infant with congenital human immunodeficiency virus (HIV) infection had immune thrombocytopenic purpura (ITP) develop at four months of age. A bone marrow aspirate had normal results in morphologic characteristics and cellularity. Flow cytometry analysis of the marrow cells showed that the predominant cell in the "lymphocyte" cluster was of B-lineage and common acute lymphocytic leukemia antigen (CALLA) positive. Southern blot analysis of marrow DNA demonstrated gene rearrangements in both the immunoglobulin (Ig) heavy chain and kappa light chain loci, confirming the presence of a clonal B-cell lymphoid proliferation. At one year of age the patient is clinically well without evidence of malignant lympho-proliferative disease. This case exemplifies a limited clonal B-cell expansion in the bone marrow of a patient with HIV infection and a benign hematologic condition.

Plasma cell malignancy in the acquired immune deficiency syndrome. Association with Epstein-Barr virus.

The development of high-grade, malignant B-cell lymphoma is a well-recognized complication of human immunodeficiency virus (HIV) infection. Plasma cell neoplasms, however, have been rarely encountered in HIV-infected people. This study presents the morphologic and immunologic features of an unusual plasma cell tumor occurring in a 31-year-old HIV-antibody-positive male. The malignancy was characterized by widespread dissemination and hypercalcemia at presentation and a clinically aggressive course. Immunoperoxidase staining of tumor tissue obtained from biopsy and at autopsy had positive results for IgM and lambda. In the patient's serum, only an IgG kappa paraprotein was detected, indicating that the tumor was nonsecretory. DNA analysis of autopsy-derived tumor tissues demonstrated clonal rearrangements of the immunoglobulin (Ig) heavy chain gene locus and rearrangements in both kappa and lambda light chain gene loci. Furthermore, DNA hybridization studies revealed the presence of Epstein-Barr virus (EBV) genomes in tumor tissue but not in nontumor tissue from this patient.

Resistance of gram-negative bacteria to antibiotics in large calf agglomerations.

The antibiotic resistance of E. coli, Citrobacter, Enterobacter-Klebsiella and Pseudomonas aeruginosa strains isolated from calves was tested. A high proportion of multiresistance was found even in E. coli strains isolated from newborn calves. Gram-negative bacteria isolated from animals in three large calfhouses were almost 100% resistant to ampicillin, tetracyclines and sulphonamides. Multiresistance was general and varied from 5 to 12 antibiotics among different strains. Initial high sensitivity to antibiotics which had never been used before was observed. Antibiotic resistance rapidly increased after use started. The usefulness of antibiotics in E. coli induced diarrhea is questioned and oral rehydration is appraised.

Impact of perceived self-efficacy in coping with stressors on components of the immune system.

This experiment examined the impact of experimentally varied perceived self-efficacy in exercising control over stressors on components of the immunological system. Immunological changes while coping with phobic stressors were measured within an intrasubject control design that included a baseline phase, an efficacy-acquisition phase, and a maximal-efficacy phase. In each of these phases, perceived coping self-efficacy, level of autonomic and endocrine activation, and several components of the immunological system were measured. Development of strong perceived self-efficacy to control phobic stressors had an immunoenhancing effect. A slow growth of perceived self-efficacy, heart rate acceleration, and cortisol activation attenuated immunological system status during the efficacy-acquisition phase. Rapid growth of perceived self-efficacy also predicted maintenance of immunoenhancement during the maximal perceived self-efficacy phase.