PASSHIV-1 treatment of patients with HIV-1 infection. A preliminary report of a phase I trial of hyperimmune porcine immunoglobulin to HIV-1.

OBJECTIVE: To study the safety of intravenously administered porcine-derived hyperimmune immunoglobulin to HIV-1, PASSHIV-1, in humans. METHODS: Fourteen HIV-1-infected individuals were treated for 5-7 days with intravenous infusions of highly purified PASSHIV-1 (> 95% pure). Two of the 14 patients were retreated 3 months later with PASSHIV-1 for an additional 5 days to evaluate side-effects from retreatment with porcine immunoglobulins. RESULTS: Ten of the patients had no side-effects from PASSHIV-1 therapy. Three patients experienced transient urticarial eruptions, which responded to antihistamine administration and did not require discontinuation of therapy. One patient, who received concomitant administration of human gammaglobulin, experienced serum sickness (type 3 hypersensitivity reaction). All patients demonstrated a significant improvement in fatigue (100% response), weight (all those with previous weight loss gained weight), fever (100% response), polyneuropathy (100% response), bronchitis (100% response), candidiasis (100% response), diarrhea (100% response), and dermatitis (100% response). One out of the five patients with Kaposi's sarcoma demonstrated > 50% improvement. Mean CD4+ cell counts in the group rose from 143 +/- 263 to 234 +/- 323 x 10(6)/l 4-6 months following completion of therapy (P = 0.013, paired Student's t-test); CD4+ counts rose > twofold in six individuals. p24 antigen, present in four patients, was negative following therapy in all patients. Other laboratory parameters that responded to therapy included: platelet counts (71% response), leukopenia (57% response), elevated lactic dehydrogenase (100% response), and elevated alkaline phosphatase (100% response). PASSHIV-1 was well tolerated by HIV-1-infected individuals. CONCLUSION: This therapy appears to be efficacious in ameliorating some of the clinical aspects and symptoms of HIV-1 infection.

Protective humoral immune responses to the human immunodeficiency virus induced in immunized pigs: a possible source of therapeutic immunoglobulin preparations.

The human immunodeficiency virus (HIV-1) induces progressive and fatal disease in infected hosts. Initially the human immune response appears to control HIV infection. This hypothesis is supported by the long latency period observed during HIV infection prior to development of the active disease state. Similarly the observation of fetal protection from HIV infection in some pregnant women who have high titered neutralizing antibody responses to the virus underscores the importance of the humoral response to HIV in limiting infection. Therefore antibody replacement therapy is likely to provide substantial clinical benefits in this and other infected populations. However, currently there is no safe source for human antibodies with the desired protective qualities necessary for passive immune therapies. For a passive immune therapy to be valid it must protect against a diverse collection of viral isolates. Such a task is likely to require a complex mixture of human antibodies or human substitute antibodies which are available in large quantities and target conserved regions of the viral envelope, such that protection from diverse isolates are realized. Porcine products have been used extensively in many human therapeutic replacement regimens. Their use is primarily due to genetic similarity of the two species at the amino acid level which results in a high acceptance of grafted prosthetics and excellent tolerance of repeatedly administered biologicals. Accordingly we have examined the immunoglobulin responses to the Human Immunodeficiency Virus (HIV-1) of the Yorkshire mixed breed pig. Immunized animals developed significant humoral immunity as judged by ELISA, Western blot, radioimmunoprecipitation, flow microfluorimetry as well as in functional assays including neutralization and syncytia inhibition. The high neutralizing activity obtained and the immunological similarity between human and porcine immunoglobulin suggests that further investigation into the use of porcine immunoglobulin as human replacement antibodies for the therapeutic treatment of HIV is warranted.

A rapid screening test for HIV-1 antibodies: application to biological studies on human tissue.

Human umbilical cord vessels are commonly used as a source of human vascular tissue for physiological studies and as a source of endothelial and smooth muscle cells. Blood samples from 236 umbilical cords were tested for the presence of HIV-1 antibodies to access the prevalence of HIV-1 infection and to evaluate possible methods for screening umbilical cords. Ten of the 236 samples were HIV-1 antibody positive by ELISA whereas 3 were positive by Western blot and a new method, the Quick-Western blot. Two of the 3 positive samples contained antibody bands against gp160, gp120, gp41, and p24 HIV-1 proteins, and one sample had antibodies against only gp160, gp120 and gp41. The Quick-Western blot required only 45 minutes for the analysis while the ELISA and Western blot took 3 hours and 18 hours, respectively. These data indicate that HIV-1 infection in mothers may present a hazard to researchers using human umbilical cords as a source of vascular tissue. The Quick-Western blot method is a simple, portable, rapid and accurate method that may be used to screen blood. The short analysis time of the Quick-Western blot allows the identification of infected blood before the tissue deteriorates as a source of cells or vascular tissue for experimental studies.

The quick western blot, a novel transportable 50-minute HIV-1 antibody test. Application in organ procurement for transplantation.

HIV-1 antibody determination by ELISA screening takes 4 hr to complete and is not reliable. Regular Western blot can take up to 24 hr. This makes organ transplantation both difficult and risky with regard to HIV-1 transmission. We developed a modification of the Western blot technique that takes under 50 min, is transportable, is definitive on site, and does not delay organ retrieval. This test has been called the Quick Western Blot. We examined 459 serum specimens from referrals; from the AIDS Testing Proficiency Panel, Walter Reed Army Institute of Research; and from 36 organ donors. All specimens were tested by ELISA HIV-1 Ab screening, the regular Western blot, and by the Quick Western Blot. The organ donors were initially tested on-site during organ procurement by the Quick Western Blot and later had complete testing by the reference methods. Compared with regular Western blot, the ELISA showed a specificity of 78.4% and a positive predictive value of 65.5%, whereas the Quick Western Blot was as reliable and specific as the regular Western blot, but much quicker. Because of the rapidity and specificity of the test, this test has particular utility in the screening of organ donors, as was shown in a case of multiple organ donation where the ELISA was negative, but the Quick Western Blot was found positive and thereby prevented the donation of HIV-1 infected organs.

Expression of c-myc and c-Ha-ras oncogenes in human lymphoblastoid cells (Namalva).

We have studied the expression of c-myc and c-Ha-ras oncogenes in this cell line. Daudi lymphoblastoid cells and normal human leukocytes served as a positive and a negative control, respectively. The Northern blot analysis using a c-myc probe revealed a 2.7 kb transcript and two larger transcripts greater than 23 kb. In the Northern blot hybridization using a c-Ha-ras probe, two transcripts with sizes of 1.8 and 6.1 kb were observed. The affinity of the Namalva RNA hybridization to c-myc probe was about 10-fold lower than that in Daudi RNA, whereas no difference in the c-Ha-ras hybridization was observed between the two cell lines. These data indicate that c-myc and c-Ha-ras are expressed in Namalva cells. It is noteworthy to consider that the difference in oncogene expression between Namalva and Daudi cells might be due to the difference in interferon properties between the two cell lines.

Antiproliferative assay of human leucocyte interferon.

An in vitro microtitration system of the antiproliferative effect of human leucocyte interferon (IFN-alpha(Le)) was investigated. The preliminary experiments suggested that the antiproliferative effect of IFN-alpha(Le) was increased by prolonging the incubation period, by reducing the target cell concentration, and might be prescribed by the total IFN amounts in the challenge medium. By employing a system consisted of 1.5 X 10(4) Daudi cells in 0.2 ml of medium containing IFN dilutions and an incubation period of 3 days at 37 degrees C, the antiproliferative effects of twenty-one lots of partially purified IFN-alpha(Le) (PIF-alpha(Le)) preparations were titrated. The interassay variations in the antiproliferative titers of three PIF-alpha(Le) established in the present system were found to be in the same range as those in the antiviral titers estimated by a standard macroplaque reduction assay. The each titration curve was parallel, and the antiproliferative titers, assessed by the reciprocals of the IFN dilutions which suppress the cell growth in 50%, were significantly (p less than 0.01) correlated to the antiviral international units of them.

Antitumor effect of human necrosis factor on human hepatoma cells PLC/PRF/5.

The antitumor activity of natural human tumor necrosis factor (TNF) on a human hepatoma cell line PLC/PRF/5 was studied in vitro. TNF produced by the LuKII human lymphoblastoid cell line showed a cytostatic effect on the hepatoma cells, whereas the growth of non-tumorigenic Chang liver cells was little affected. The combined effects of TNF and interferon-gamma (IFN-gamma) were additive on the PLC/PRF/5 cells as shown by statistical analyses. The same combination showed synergistic effects on a human breast cancer cell line BT-20, which was highly sensitive to TNF. These data may provide some informations concerning the use of TNF in the treatment of hepatoma.

Oncogene expression in human hepatoma cells PLC/PRF/5.

The expression of 7 cellular oncogenes in a human hepatoma cell line PLC/PRF/5 was studied using Northern blot analyses. Among the oncogenes tested, c-abl, c-fes, c-fms, c-myc, c-Ha-ras and c-sis were expressed. The oncogene c-Ki-ras was not expressed. The length of the mRNAs expressed was almost consistent with published data. Compared to the oncogene expression in Daudi lymphoma cells, the same kind of oncogenes were expressed in PLC/PRF/5 cells, but the intensity of the signal in each oncogene expression was stronger in Daudi cells than in PLC/PRF/5 cells. Considering the cellular localization and the function of each oncogene, the oncogene survey in hepatoma cells broadens the knowledge of hepatocarcinogenesis and the character of human hepatoma cells.

Comparison of the antitumor effects of human natural and recombinant interferons (alpha and beta) on human cancer cell lines.

The antitumor effects of 5 different subtypes of human type I interferons (IFN-alpha(Le), rIFN-alpha A, rIFN-alpha D, IFN-beta, and rIFN-beta) were studied on 3 human cancer cell lines (Daudi lymphoma, PLC/PRF/' hepatoma, and G361 melanoma). IFN-alpha(Le) was the most potent against Daudi cells. The effects of IFNs were cytostatic. On the other hand, 500 IU/ml of IFN-alpha(Le), IFN-beta and 5000 IU/ml of rIFN-beta showed cytocidal effect against PLC/PRF/5 cells. The G361 cells were the least sensitive to the IFNs tested. This is the first report on the antitumor effect of rIFN-beta isolated from insect cells. The effect of this rIFN-beta was similar to that of IFN-beta.

A cytotoxic substance (CTS-51) produced by human buffy coat cultures stimulated by staphylococcal enterotoxin B: further characterizations and combined action with interferon.

A recently recognized unique cytotoxic substance, CTS-51, was tested for the hear or acid stability, trypsin digestion and dialysis. Moreover, influences of elevated incubation temperatures or serum concentrations of medium on the cytotoxic activity of CTS-51, and the combination effects of CTS-51 and human leucocyte interferon (HuIFN-alpha (Le)) were investigated. The cytotoxic activity of CTS-51, which is promoted by a small molecule easily passable the dialysis membrane, was found to be very stable to heat (even at 100 degrees C for 30 min) or acid (pH 2.0 for 24 hr at 4 degrees C) treatments. The treatment with 0.75% trypsin for 1 hr did not diminish the CTS-51 activity. The susceptibility of Daudi lymphoma cells to the antiproliferative action of HuIFN-alpha (Le) was further potentiated by treating the cells with CTS-51 for 16 hr. On the other hand, the CTS-51 activity which was revealed to be prescribed by its concentration in the medium, was not potentiated at 39 degrees C when compared to that at 37 degrees C in contrast to HuIFN-alpha (Le) action, and was reduced according to the increase of the fetal calf serum concentration in the medium.

Effects of cimetidine on various biological activities of human leucocyte interferon and on interferon production in lymphocytes.

In vitro effects of cimetidine on the antiviral, the antiproliferative and the natural killer (NK) cell stimulating activities of human leucocyte interferon (IFN-alpha (Le], and on the Sendai virus induced interferon production in lymphocytes were investigated. The antiviral activity of IFN-alpha (Le) was dosedependently enhanced by cimetidine, which alone did not demonstrate any antiviral action. The observed titer of the same IFN preparation titrated with 60 micrograms/ml of cimetidine was significantly higher (p less than 0.05) than that without cimetidine. Cimetidine slightly but dosedependently inhibited the growth of Daudi or G-361 cells. The combination effects of these two agents were revealed to be statistically additive or synergistic. The NK cell activity of peripheral blood lymphocytes (PBL) was suppressed after a 16 hr pretreatment with cimetidine. Furthermore, the interferon production in normal lymphocyte cultures was suppressed dosedependently by cimetidine. On the other hand, the augmentation of the NK cell activity by interferon was further potentiated by cimetidine.

Cimetidine enhances interferon induced augmentation of NK cell activity and suppresses interferon production.

Effects of cimetidine on the augmentation of natural killer (NK) cell activity by human leukocyte interferon (Hu IFN-alpha (Le] and on the IFN production from donor leukocytes were investigated in vitro. NK cell activity of PBL was suppressed after 16 hours pretreatment with 0.3, 3.0, and 30.0 micrograms/ml of cimetidine. On the other hand, there was dose dependent suppression of Hu IFN-alpha (Le) production in PBL by cimetidine. The NK cell activity of PBL augmented by Hu IFN-alpha (Le) was further enhanced when the effector cells were pretreated with cimetidine or when cimetidine was added together with IFN. K562, G361, and PLC/PRF/5 cells were used as target cells. Our results suggest that a 16 hour treatment with cimetidine alone may suppress NK cell activity of PBL, probably due to reduced IFN production by leukocytes. On the other hand, cimetidine may enhance the IFN induced NK augmentation in PBL in vitro.

Antiviral and antiproliferative activities of human leukocyte interferon potentiated by cimetidine in vitro.

The influence of cimetidine on antiviral activity of leukocyte interferon (IFN-alpha (Le] was studied in plaque-reduction assays using Utrecht (U) amnion cells challenged with vesicular stomatitis virus (VSV) and in CPE inhibition assays using A549 cells challenged with encephalomyocarditis (EMC) virus and WISH cells challenged with VSV. The IFN-alpha (Le)-induced antiviral activity was slightly enhanced in cells treated with cimetidine, whereas cimetidine treatment alone did not show any antiviral effect. The observed titer (OT) was significantly higher (p less than 0.05) in cells treated with cimetidine together with IFN-alpha (Le) compared with the control without cimetidine. The effect of cimetidine on IFN-alpha (Le)-induced cell growth inhibition was studied on Daudi (a Burkitt's lymphoma cell line) and on G361 (a melanoma cell line) cells. The growth of these cells was slightly suppressed by cimetidine alone. When cells were treated with IFN-alpha (Le)/cimetidine, the cell growth inhibition rates were significantly higher (p less than 0.02) than the rates obtained with IFN-alpha (Le) or cimetidine alone. These results indicate that cimetidine can enhance the antiviral as well as the antiproliferative activities of IFN-alpha (Le) in "in vitro" studies.

Biological and immunological properties of native species of human leukocyte interferon.

The successful purification of the native species of human leukocyte interferon (Hu IFN - alpha) has made it possible to perform a detailed biological immunological analysis which has been disclosed that the human interferon alpha species are represented by 13 individually separated species having molecular weights in the range of 16.6 to 33.5 kD (1-3). The stained interferon proteins were eluted individually from the gel slices and assessed for antiviral activity in the human, monkey and bovine cells, as well as for immunomodulatory effect in vitro on human lymphocytes. Interferon titrations were performed in human and in bovine cells and it was disclosed that one interferon species did not have any activity in the human system, at all (2). In contrast to the human activity, the bovine activity in each Hu IFN - alpha species increased along with decreasing molecular weights. Based on equal amounts of human interferon units, the "immunological efficacy" of the 13 different human IFN alpha species yielded the following in cellular immune systems (4): 1) potentiation of the natural killer cell (NK) function was a property of all species, although the two lower species (16.6 kD and 16.9 kD) were clearly less efficient having "titers" in the NK system reduced 25-fold; 2) enhanced expression of HLA on lymphocyte membranes was induced by all of the Hu IFN alpha species to the same extent; and, 3) addition of interferon to mixed lymphocyte reaction (MLR) augmented the cytotoxic mediated lympholysis (CML) outcome of the cultures. In this system all 13 species exerted their effort equally well.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature influences on different human alpha interferon activities.

Influences of different incubation temperatures within the physiological range on three different biological activities of human leukocyte-derived alpha interferon (IFN) (the antiviral effect, the antiproliferative activity, and the augmentation of natural killer cell (NK) activity) were investigated in vitro. Using the plaque-reduction assay (U cells challenged with VSV), the antiviral activity by IFN was found to be lower at 35 degrees C and higher at 38 degrees C and 39 degrees C than at 37 degrees C. Using the CPE (cytopathogenic effect) inhibition technique (Vero cells challenged with VSV), the antiviral activity was slightly enhanced at 38 degrees C, 39 degrees C, and 40 degrees C, respectively, when compared with 37 degrees C. The antiproliferative activity on Daudi cells and G-361 melanoma cells was enhanced at elevated temperatures. On the other hand, the antiproliferative activity on PLC/PRF/5 hepatoma cells was lower at 38 degrees C and 39 degrees C than at 37 degrees C, but higher at 40 degrees C. NK activity of PBL after 2 h incubation at 41 degrees C was remarkably lower than that at 37 degrees C, while it was not affected by 2 h incubation at 35 degrees C and 39 degrees C, respectively. When PBL was treated with IFN for 2 h at the temperatures described above, NK activity was equally augmented at all temperatures tested. Our results suggest that elevated incubation temperature potentiates the antiviral and the antiproliferative activities, but does not affect the NK augmenting activity of HuIFN-alpha (Le).

Treatment of dysplasia of the cervical epithelium with an interferon gel.

Human leukocyte interferon was incorporated into a hydrophilic gel and applied to the cervix of six patients suffering from moderate to severe dysplasia or carcinoma in situ of the cervix. Only patients showing no spontaneous regression during at least 27 consecutive weeks were admitted to the trial. The diagnoses were established by Papanicolaou smears and biopsy specimens of the cervix. The gel was applied directly on the cervix twice weekly for six weeks, and only minor clinical improvements were seen. However, after an additional six weeks, six of six patients responded positively; three of them regressed completely. No side effects were noted.

Interferon therapy in neoplastic disease. A preliminary report.

The treatment of 10 patients having neoplastic disease with exogenous i.m. interferon therapy is described. The interferon given is partially purified interferon produced from human leukocytes. Sendai virus is used as interferon inductor. The patients reported in this paper have been on treatment for periods of 2-28 months. Apart from initial periods of fever, no side-effects have been recorded. Patients suffering from bladder papillomas have shown partial regression after a few months of therapy. The other cases treated are too few to warrant any conclusions, but the therapy does seem to have been beneficial.

The relationship between pregnancy, HCS and B lymphocytes.

The quantitative distribution of the lymphocyte subpopulations (B, T and null) and the serum concentrations of Human Chorionic Somatomammotropin (HCS), immunoglobulins IgG, IgM and IgA, complement component C4 and C1 inactivator, were estimated in venous blood samples from 32 women at various stages of pregnancy and compared with a control series of 7 non-pregnant normal women. A significant decline in the B cell percentage during pregnancy was seen. The fall in the percentage of the B lymphocytes was found to be concomitant with the rise in HCS concentration. No significant changes in the other parameters studied were present.

B, T and null lymphocytes in newborn infants and their mothers.

Estimation of B, T and null cells were performed on 29 newborn healthy babies and 16 mothers. The lymphocytes were isolated from peripheral venous blood, which is considered to be more representative of the immune state in the newborn than the cord blood. B lymphocytes were estimated by cytofluorometric measurements, T lymphocytes by sheep red blood cell rosette technique, (SRBC-R). Combined immunofluorescence and SRBC-R technique revealed the null cells. In the newborn babies the amount of B and T cells were found to be diminished. In the mothers the amount of B lymphocytes were low compared with normal adults. The rather high null cell percentage found in the babies might represent immature precursor cells. Mothers seem to be immuno-depressed as reflected in the low amount of B cells.