Immunostimulatory human urinary protein.

Abstract

We have previously extracted a protein from inflammatory mouse granuloma that fully protects normal mice against lethal Listeria monocytogenes infection. We now report that polyclonal antibodies directed against this protein react with a human urinary fraction that also provides full protection of normal mice against L. monocytogenes. Murine monoclonal antibodies completely inhibit the protective activity of the human urinary fraction. We have purified to apparent homogeneity a human glycoprotein of 43 kDa (HGP.43), pI = injection of C57BL/6 mice with Bio-Gel P-100 (Bio-Rad), the gel was withdrawn and the inflammatory cells were recovered by filtration through a 400-mesh screen. The cells were centrifuged (250 x g for 10 min), washed twice with ice-cold Dulbecco's modified Eagle's medium (DMEM), incubated (1 x 106 cells per ml, 0.5 ml per culture tube) for 24 hr, and washed to remove polymorphonuclear cells. Lewis tumor cells (3) were isolated from murine lung metastases and cultured in plastic Petri dishes in DMEM containing 10% (vol/vol) fetal calf serum at 3TC in an atmosphere of 7.5% C02/92.5% air.Antibodies. Polyclonal rabbit anti-mouse granuloma protein antibodies were prepared as described by Fontan et al. (1). Polyclonal antibodies against the human urinary protein were raised in rabbits by s.c. injection of 10 ,ug in Freund's complete adjuvant and twice-monthly booster injections. Immunoglobulins were purified by precipitation in (NH4)2SO4 at 33% saturation and DEAE-gel chromatography. Antibodies against human al-AGP were purified by means ofimmunoaffinity chromatography using pure a1-AGP (Sigma) coupled to CNBr-activated Sepharose. Monoclonal antibodies against the human urinary protein were prepared as follows. Female BALB/c mice were immunized by s.c. injection of 50 jug of a semipurified active human urinary fraction. Spleen cells were fused with the SP2/0 BALB/c myeloma cell line. Wells with hybrid growth were screened for reactivity with the urinary fraction by using an ELISA. Positive wells were subcloned twice by limiting dilution. One of the subclones (K52.5G2) was chosen for the neutralization test. This clone produces an IgG1 that was purified by (NH4)2SO4 precipitation and DEAE-gel chromatography. The supernatants and antibody-free controls were tested in mice challenged with a lethal inoculum ofL. monocytogenes.All the materials, solutions, and tissue culture media were free of endotoxin.Statistics. Results are expressed as the mean ± SEM. The significance of the differences between experimental groups was analyzed by Student's t test. A P value of <0.05 was considered significant. RESULTS Purification and Characterization of the Urinary Protein. After Amicon ultrafiltration concentration of the urines, the material was chromatographed on a DEAE-Sephacel anionexchange column and eluted with 0.2 and 1 M NaCl. All the immunostimulating activity was eluted with 0.2 M NaCI (Fig. 1). The active fractions were pooled, dialyzed, and loaded onto a Cibacron blue column to remove contaminat...

A 92-kDa human immunostimulatory protein.

“…This glycoprotein, purified to homogeneity, protected mice against Listeria infection. This increased resistance was also found in scid mice (4). In vitro, HGP.43 induced cytotoxic activity in macrophages against Lewis carcinoma cells.…”

“…We have previously shown that two proteins of 43 and 92 kDa isolated from human urine protected mice against a lethal inoculum of L. monocytogenes (4). After Pronase treatment of the 92-kDa protein, all immunostimulatory activity was associated with the 43-kDa protein (HGP.43).…”

“…Twenty-four-hour urine specimens were collected from healthy male and female human beings and stored at -200C as described by Fontan et al (4). THP was isolated from pooled urine after two salt precipitations as described by Tamm …”

Randomized phase II clinical trial of chemo-immunotherapy in advanced nonsmall cell lung cancer

“…These facts concur with the reported immunomodulatory activity of the IAS components: Cyclophosphamide, low dose, 3 days before antigen stimulation, decreases the CD4+CD25+ (T-reg) cell population (Berd et al 1982; Ghiringhelli et al 2004). The magnesium silicate granuloma has been demonstrated as a remote enhancer of systemic macrophage activity committed to the protective responses (Fauve and Hevin 1977; Fontan et al 1983, 1992; Fauve et al 1987). GM-CSF, subcutaneously, administered around the antigenic stimulation, recruits and activates the antigen presenting cells, mainly dendritic cells, allowing a stronger immune response (Disis et al 1996; Dranoff 2002).…”

“…This loco-regional immunomodulation is decisional because it elicits a systemic protective lymph nodes conditioning. Among other agents (erythrocytes or other local inflammatory agents) to create an ITS, magnesium silicate that produces a subcutaneous granuloma (MSG) was reported as a strong inducer of remote macrophage activation with enhancement of protective antitumor immune responses, when it is performed during the 4 days previous to the antigen (Fauve and Hevin 1977; Fontan et al 1983, 1992; Fauve et al 1987). It was also reported that granulocyte macrophage-colony stimulating factor (GM-CSF), injected subcutaneously, simultaneously or around the time of antigen stimulation, is a recruiter and activator of antigen presenting cells, mainly dendritic cells (Disis et al 1996; Dranoff 2002).…”