Alkaline phosphatase band-10 fraction as a possible surrogate marker for human immunodeficiency virus type 1 infection in children.

We report the utility of a possible lymphocyte fraction of alkaline phosphatase (ALP band-10) activity in serum to predict human immunodeficiency virus type 1 (HIV-1) infection in children born to HIV-1-seropositive mothers. The presence of ALP band 10 in serum consistently correlated with HIV-1 infection status as judged by positive HIV-1 culture, two consecutive HIV-1 p24 antigen results greater than 30 pg/mL in serum, and the subsequent confirmation of seroconversion to HIV-1 antibody after clearance of maternal IgG anti-HIV-1 antibody ascertained between 15 to 24 months post partum. Infection with HIV-1 was correctly identified in 31 samples from 18 patients ranging in age between 0.1 to 10 years; the absence of similar infection was noted in 14 samples from nine patients who served as controls and whose serum samples did not exhibit ALP band-10 activity. This ability of serum ALP band-10 activity to predict HIV-1 infection status in children as young as 2 months may be useful as a surrogate marker for early identification of HIV-1 infection in infants born to HIV-1-seropositive women long before the clearance of maternal anti-HIV-1 antibodies can be ascertained.

A rapid test for the detection of human immunodeficiency virus antibodies in cord blood.

A commercially available rapid test (HIVCHEK) was compared with an enzyme-linked immunosorbent assay (ELISA) for identifying human immunodeficiency virus type 1 in the serum of newborn infants. Of 1309 cord blood samples tested, the HIVCHEK test detected all the true-positive samples detected by ELISA. Of the 35 samples with positive ELISA results, six had negative results on Western blot; only 1 of the 30 samples with positive HIVCHEK results had negative results on Western blot. Thus the HIVCHEK test can be used to facilitate the rapid identification of HIV-1 in the serum of newborn infants.

GM-CSF induces eosinophilic cell growth-promoting activity on human fetal liver cells.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) has been described as a multi-lineage growth factor that induces in vitro colony formation of bone marrow erythroid burst-forming units (BFU-E), multipotential colony-forming units (CFU-GEMM), granulocyte-macrophage CFU (CFU-GM), granulocyte CFU (CFU-G), macrophage CFU (CFU-M), as well as eosinophil colony-forming units (CFU-Eo). Because of the preeminent role of the liver in fetal hematopoiesis, the effect of human recombinant GM-CSF (hrGM-CSF) on hematopoietic cells isolated from human fetal liver was tested in liquid cultures and in semisolid colony assays. hrGM-CSF induced a significant increase in the number of mature eosinophils in liquid culture and to a lesser extent in semisolid cultures when compared to untreated culture controls. The kinetics of this effect on eosinophils reached its peak on day 21 of culture. When GM-CSF and erythropoietin (Ep) were added simultaneously to the cultures, no significant change in the number of eosinophils compared to hrGM-CSF alone was observed. Ep or granulocyte colony-stimulating factor (G-CSF) did not show any CFU-Eo activity when added separately or simultaneously to both liquid and semisolid cultures. These results indicate that hrGM-CSF alone may be a potent stimulating factor for CFU-Eo obtained from human fetal liver and, in combination with other growth factors, control optimal development of human fetal eosinophils.

Inhibition of HIV-1 infection by alkylureas.

The risk of infection by HIV-1 through transfusion of contaminated blood products has been markedly decreased but not eliminated by serological screening of donors. Methods are required to further minimize or eliminate the risk of infection of blood product recipients. We therefore examined the capacity of alkylureas to inhibit infectivity of HIV-1. Incubation of free HIV-1 virions with alkylureas suppressed their infectivity, and the minimal inhibitory concentration of the alkylureas was related to the length of the alkyl chain. Butylurea, the most potent inhibitor of HIV-1, inhibited the infectivity of 10(5) median tissue culture infective dose (TCID)50 of HIV-1, chronically HIV-1-infected H9 cells and mononuclear cells from two HIV-1-infected patients. Size fractionation of HIV-1 following incubation with butylurea indicated that the structure of the virus was disrupted by butylurea. This study demonstrates that butylurea, at a concentration that has been shown not to affect red blood cell function, can inhibit infectivity of extracellular and intracellular HIV-1. Since the HIV-1 inhibitory capacity of the alkylureas increases with the length of the alkyl side chain, it is likely that hydrophobic interactions between the alkylureas and HIV-1 are responsible for the observed effect.

Human immunodeficiency virus (HIV) circulating immune complexes in infected children.

Circulating immune complexes (CIC) were studied for the presence of human immunodeficiency virus (HIV) antigens (HIV-Ag) in 55 children infected by the human immunodeficiency virus type 1 (HIV-1). CIC were elevated in 85% of patients. In 33 of 55 patients CIC included at least one HIV-Ag (HIV-Ag-CIC). Sixty percent of patients had p17 antigen, 50% had p24 antigen, and 16% had gp120 associated with CIC. Levels of HIV-Ag-CIC did not correlate with free serum HIV antigens. Patients with high HIV-Ag-CIC had a more severe clinical course and 90% of those with markedly elevated HIV-Ag-CIC (greater than 3+) have died within 6 to 24 months. HIV-Ag-CIC were also present in some patients including neonates and young infants in whom free HIV-Ag was undetectable. Monitoring of HIV-Ag in isolated CIC may be of value for early detection of HIV infection and for monitoring of disease outcome.

Vertical transmission of human immunodeficiency virus is correlated with the absence of high-affinity/avidity maternal antibodies to the gp120 principal neutralizing domain.

Many, but not all, infants born to mothers infected with the human immunodeficiency virus (HIV) are infected in utero. We have now shown that mothers who have high-affinity/avidity antibodies directed toward the principal neutralizing domain (PND) of gp120 are less likely to transmit HIV to their children. An ELISA that preferentially measures the level of the biologically functioning, high-affinity/avidity antibodies against PND is described. In a retrospective study of 15 maternal/neonatal serum samples, the assay correctly identified the 4 uninfected and the 11 HIV-infected infants. Other clinical and laboratory parameters such as p24 antigen, phytohemagglutinin mitogenic index, and absolute surface antigen T4+ cell counts did not accurately predict HIV fetal transmission. In addition to introducing a promising diagnostic tool, this study provides the in vivo evidence that protective antibodies may prevent infection by HIV.

Human suppressor factors constitutively produced by T-T cell hybridomas: functional and biochemical characterization.

We have recently developed a new method (Hybridoma 6:589, 1987) for the generation of human T-T cell hybrids. This method is based on a new selection procedure that involves cloning the hybrids in soft agar, screening by HLA-typing or appropriate functional tests and recloning by limiting dilution. T-T cell hybrids were separated from the parent line on the basis of their ability to form colonies in soft agar, whereas the parent lymphoblastoid T cell lines did not. HAT medium was not used in our selection procedure. Using this method, we have succeeded in developing human T-T cell hybrids (as determined by HLA-typing) constitutively producing B cell growth factor (BCGF) (Hybridoma 6:589, 1987) or suppressor factors. These hybrids were obtained by fusing MLC or Con A T cell blasts with cells from the Molt 4 or Jurkat lymphoblastoid T cell lines. T-T cell hybridomas, derived by fusing Con A-stimulated lymphocytes with cells from the Jurkat T cell line, produced suppressor factors inhibiting: (1) proliferative response in vitro of human peripheral blood mononuclear leukocytes to mitogens and to allogeneic cells in mixed lymphocyte culture; and (2) immunoglobulin synthesis and secretion by mononuclear leukocytes in the PWM-induced differentiation system in vitro. A suppressor factor with these inhibitory properties was also identified in supernatants of the Jurkat T cell line. These suppressor factors were ammonium sulphate precipitable, pH 2 labile, non-dialyzable and they were inactivated by treatment at 56 degrees C for 30 minutes. They exhibited a molecular weight in the range of 50,000-70,000, as determined by gel filtration, and were not gamma or alpha interferon or lymphotoxin/TNF. They did not lyse human lymphoblastoid tumor cell lines nor did they affect the viability and cell numbers of human mononuclear cells even after prolonged incubation (88 hr). They appeared to be cytostatic rather than cytotoxic molecules. The Jurkat suppressor factor is different from those produced by the hybrids on the basis of: (a) different isoelectric points; and (b) the ability of the Jurkat factor to arrest proliferation to PHA of human mononuclear cells in the S phase, whereas the 160 and 169 factors arrest proliferation at the G1 phase of the cell cycle. Certain of these suppressor factors (produced by the hybrids 153, 160, 170, and the Jurkat T cell line) also inhibited proliferative responses of mouse lymphocytes in vitro. In contrast, suppressor factors produced by the 169 and 77 hybrids did not inhibit any murine responses.

Hybridoma-derived human suppressor factors: differential effects on mouse lymphocytes.

We have recently identified a family of suppressor factors produced by certain human T-T cell hybridomas that we developed (references 1 and 2) and by the Jurkat T cell line. These suppressor factors significantly inhibited proliferative responses to mitogens and allogeneic cells in mixed lymphocyte culture and antibody production by human peripheral blood mononuclear cells. We investigated and report here the effect of these suppressor factors on certain in vitro murine immune responses. Suppressor factors produced by certain of these hybrids, such as 153, 160, 170 and by the Jurkat T-cell line were able to inhibit: (1) proliferative responses to mitogens of mouse thymocytes and splenocytes; (2) proliferative responses of mouse splenocytes to allogeneic cells in mixed lymphocyte cultures; (3) primary in vitro antibody responses of mouse spleen lymphocytes to sheep erythrocytes; (4) primary in vitro antibody responses of mouse spleen lymphocytes to a T-cell independent antigen (TNP-Ficoll). Inhibition of murine immune responses in vitro by these suppressor factors was regular and reproducible and it was observed in a large number of experiments. In contrast, suppressor factors produced by the 169 and by the 77(38F3) hybrids did not suppress the murine immune responses. The basis for these differences are not known at the present. The ability of human suppressor factors to inhibit effectively mouse immune responses provides an additional opportunity for the characterization of the properties of these factors in vivo using mouse models of human disease.

A new method for the development of human T-T cell hybrids without the use of HAT medium.

We report here a new method for the development of human T-T cell hybrids by fusing mitogen- or alloantigen-stimulated T cells with non-mutagenized cells from human lymphoblastoid T cell lines. This method is based on a new selection procedure where the hybrids are separated from the parent T cell line on the basis of their ability to form colonies in soft agar. In contrast, cells from lymphoblastoid T cell lines Molt-4 and Jurkat do not form colonies in agar. Hybridoma colonies are retrieved from the agar plates, expanded in culture, screened by HLA-typing and appropriate functional tests and recloned several times by limiting dilution. HAT medium, which contains thymidine that appears to be toxic to the hybrids, is not used in our selection procedure. Using this method, we developed human T-T cell hybridomas (as determined by HLA-typing) producing B-cell growth factor (BCGF) either constitutively or after induction with Concanavalin A (Con A). Certain other T-T cell hybrids produced suppressor factor, significantly inhibiting proliferative responses of human peripheral blood mononuclear leukocytes to PHA, Con A and allogeneic cells in mixed lymphocyte culture.

Leukocyte subpopulations elicited by a nontumorigenic variant of B16 melanoma: their role in direct rejection of the melanoma and in prevention of tumorigenesis in Winn assays.

The mechanisms by which various leukocyte subpopulations elicited by an immunogenic, nontumorigenic subclone (C3471) of B16 melanoma caused rejection of the tumorigenic parental melanoma (B559), were investigated. Leukocytes from C3471-immune mice were co-injected with B559 tumor cells in Winn assays into normal syngeneic recipients. Tumor formation by B559 cells was prevented when C3471-immune (a) unfractionated peritoneal leukocytes, or (b) glass-adherent peritoneal cells (90% macrophages), or (c) nylon wool purified nonadherent cells (95% Thy-1.2+) were used in the Winn assays. If the C3471-immunized mice were treated with antithymocyte serum before harvest of their peritoneal cells, none of these leukocyte populations were effective in the Winn assay. However, macrophages from these immunologically compromised donors regained their tumoricidal activity after incubation in vitro with T lymphocytes from untreated C3471-immune donors; similarly, C3471-immune lymphocytes rendered normal resident peritoneal macrophages tumoricidal in Winn assays. When C3471-immunized mice were irradiated or treated with antithymocyte serum before direct challenge with B559 cells, melanomas developed, thus providing additional evidence for the need for intact T cell function to establish immunity against the melanoma. Furthermore, when Winn assay recipients were treated with antithymocyte serum, neither C3471-immune macrophages nor T cells were able to prevent tumor formation. These findings indicate that antithymocyte serum-sensitive (Thy-1.2+) lymphocytes are necessary both for the generation of tumoricidal leukocytes in C3471-immunized mice, and for the rejection of B559 melanoma by demonstrably tumoricidal macrophages in Winn assay recipients. In addition, long-lasting immunity developed in 50% of the normal mice that had received both C3471-immune peritoneal cells and B559 tumor cells, as manifested by their capacity to reject a second challenge with B559 cells 40-60 d later.

Relationship between rejection of several syngeneic tumors and retrovirus production by 5-bromodeoxyuridine-grown melanoma cells: lack of protection in natural killer-deficient beige mice.

Bromodeoxyuridine-grown B16 melanoma cells (C3471) immunize mice against not only the parent melanoma but also two other C57BL/6 tumors: a mammary adenocarcinoma and a methylcholanthrene-induced sarcoma. We have shown that the endogenous retrovirus induced in C3471 cells by bromodeoxyuridine can persistently infect feral mouse (SC1) cells and that they then become as efficient as C3471 cells in preventing tumors. Uninfected SC1 cells cannot protect. Neither C3471 nor virus-infected SC1 can prevent B6MS5 or B6MS7, two other non-virus-producing sarcomas, from forming tumors. Meth 4, mammary adenocarcinoma, and L-cells produce retrovirus and prevent melanoma formation in half the mice challenged. Significantly, C57BL/6 mice homozygous for the beige mutation are unable to reject melanoma challenge after C3471 immunization, although their normal littermates do so efficiently. We conclude that production of retrovirus is in some way responsible for the cross-reactive immunizing capacity of C3471 cells and that cells in which the beige mouse is deficient play a role in the rejection process. Beige mice have been shown to be deficient in natural killer cells and abnormal in macrophage kinetics. In addition, C3471-induced protection against B559 melanoma appears to involve host cells sensitive to anti-thymocyte and anti-lymphocyte sera. We hypothesize that the retrovirus-producing cells may cause induction of interferon, which augments the cytocidal activity of natural killer cells and macrophages, killing sensitive tumor cells.

Macrophages elicited with heat-killed bacillus Calomette-Guérin protect C57BL/6J mice against a syngeneic melanoma.

We have demonstrated that a murine cytotoxic peritoneal cell can be elicited by intraperitoneal immunization with heat-killed Mycobacterium bovis, strain Bacillus Calmette-Guérin (BCG). When these cells are injected together with cells of clone B(5)59 of B16 melanoma in a Winn-type transfer assay into syngeneic C57BL/6J mice, the tumorigenic potential of the melanoma is completely abrogated. Similarly, mice immunized intraperitoneally with dead BCG are protected against intraperitoneal challenge with a number of B16 melanoma cells sufficient to cause tumors in 100% of control mice. However, mice immunized intraperitoneally with dead BCG are not protected against tumor formation when B16 melanoma cells are injected subcutaneously. Co-injection of BCG-elicited peritoneal cells with B16 melanoma cells into nude or sublethally irradiated (650 rad) mice inhibits tumor formation in > 85% of the mice, indicating that additional participation of host bone marrow- or thymus-derived leukocytes is not required to eradicate the tumor implant. The effector cell in the BCG-induced peritoneal exudate is adherent and phagocytic and is a mononuclear phagocyte. Nonadherent lymphoid cells from the same BCG-induced peritoneal exudate and from thioglycollate-broth-elicited granylocytes and macrophages neither prevent nor delay B16 tumor formation.