A two-stage screening test for pregnancy-induced hypertension and preeclampsia

PURPOSE: To define the inductive pathways leading to rat tear IgA antibody responses. METHODS: Fluoresceinated dinitrophenylated bovine serum albumin was encapsulated in poly(lactide-co-glycolide) microparticles and was administered by intranasal, ocular topical, or gastrointestinal routes. Histologic methods were used to determine the microparticles' ability to access tissues associated with mucosal inductive pathways. Rats were immunized with microencapsulated antigen by intranasal or ocular topical routes. Tear IgA and serum IgG antibody concentrations were assessed by radioimmunoassay. The frequency of antibody-secreting cells in tissues, postulated to function in tear IgA induction, was measured by enzyme-linked immunospot assay. RESULTS: Although uptake of microencapsulated antigen was greatest at the site of delivery, ocular topical administration resulted in antigen uptake in the conjunctiva and in nasal-associated lymphoid tissue. Intranasal immunization resulted in earlier and significantly higher tear IgA and serum IgG antibody responses and in higher frequencies of antibody-secreting cells in corresponding draining cervical lymph nodes and lacrimal glands than did ocular topical immunization. CONCLUSIONS: Nasal-associated lymphoid tissue functions as a primary inductive site for tear IgA antibody responses by contributing triggered IgA-committed B cells to the lacrimal gland.     

Immunopathology of the uveitis in canine leishmaniasis

Particular immunopathological features and their effects on the vascular permeability of different ocular structures were analysed in two dogs naturally infected by Leishmania infantum. The existence of specific anti-Leishmania immunoglobulin G (IgG) in the aqueous humour was confirmed by the ELISA technique. There was no correlation between antibody levels in the aqueous humour and the related serum. The histopathological study of the eyes showed the existence of lesions in various ocular structures. The ciliary processes, ciliary body, sclerocorneal limbus, iris and lacrimal duct showed intense inflammatory zones with lymphocyte infiltrates, plasmatic cells and macrophages with amastigote forms of Leishmania. In addition vasculitis with dilation and thrombi were also detected in both cases, with consequent oedema and hyalinization. The immunohistochemistry analysis revealed the presence of granular and diffuse IgG deposits in the ciliary body, ciliary processes, sclerocorneal limbus and iris. Furthermore, numerous thrombosed vessels were observed in the sclerocorneal zone and iris. Complement 3 (C3) fraction deposits were not present in the ocular structures. The present data suggest that the ocular lesions may have an immunopathological origin.     

Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyer's patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.     

Serological diagnosis of experimental Enterococcus faecalis endocarditis

A modified rat model of endocarditis with catheterization for 2 days was established in female Lewis rats using different inocula of Enterococcus faecalis (strain no. EF 19) in order to measure IgG antibodies in serum during the course of infection. Increasing the inocula intravenously resulted in an increase in the CFU/g vegetation and the CFU/g spleen, the ID50 being about 10 CFU/ml and the ID90 about 1x10(2) CFU/ml. The lowest bacterial inoculum infecting 100% of the rats was 3x10(3) CFU/ml, and for further investigations we used this inoculum size. Rats were sacrificed on day 2, 5, 7, 9, 11 and 28 after infection. The CFU/g vegetation and the CFU/g spleen increased until day 7 and then decreased. Serum samples were collected from 129 rats at different times after challenge. Three different ELISA systems were established to measure the IgG antibody responses: E. faecalis sonicate ELISA (a pool of four sonicates of strain no. EF 10, EF 11, EF 19 and EF 48), E. faecalis whole cell ELISA (strain no. EF 19) and E. faecalis purified cell wall ELISA (strain no. EF 19). An IgG antibody response was detected already on day 2, and except for a minor decrease on day 6/7 the antibody response continued to increase until day 14 (whole cell ELISA and sonicate ELISA) and day 21 (purified cell wall ELISA) when a plateau was reached. Significant increases in IgG antibody responses (p<0.05) were found between groups of rats from days 0-2, 2-8/9 and 8/9-14 in the E. faecalis whole cell and sonicate ELISAs and from days 0-2, 2-10/11 and 10/11-21 in the E. faecalis purified cell wall ELISA. In conclusion, we established a model of endocarditis in rats with catheterization for 2 days and were able to demonstrate an increase in IgG antibodies during the course of infection.     

Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA

A lysate of human herpesvirus type 6 (HHV6) infected HSB2 cells was used as antigen for an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibody to HHV6. 78 clinical samples were tested for the presence of HHV6-specific IgM. Nine specimens, all from children under 4.5 years of age, were found to be reactive indicating probable acute infection with HHV6. Sera from 12 healthy adult blood donors and from 88 of 90 adults over the age of 35 with unspecified health conditions tested negative for HHV6 IgM, indicating a minimum specificity estimate of nearly 98% in these patients. Cross-reactivity of antibody to other herpes viruses with the HHV6 ELISA antigen was not detected. Six hundred and ninety-six serum samples from individuals of different age groups were examined for IgG antibody status. In 94% of these samples, IgG antibody was detected. Our data suggests that most Canadians possess antibody to HHV6 by 1 yr of age and that on average, antibody levels remain high through early adulthood but begin to decline with advancing age. The ELISA described is a reliable test for the measurement of IgG and IgM antibodies for both clinical diagnosis and epidemiological studies.     

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay for specific IgG antibody produced in vitro

IgG antibody production by immune spleen cells in vitro was assayed by antibody-dependent cell-mediated cytolysis (ADCC). The use of this technique as a sensitive and reproducible alternative to indirect haemolytic plaque assays is examined.     

Long-term lipid-based total parenteral nutrition activates mononuclear cells and modulates membrane lipid composition in pigs

This study aims to assess the possible strain-dependent variations in detection of Toxoplasma antigens and antibodies. The virulent RH strain or avirulent Beverley strain of T. gondii were injected into mice, intraperitoneally, and their antigens, antibodies and parasites were identified from the blood or tissues; liver, brain and spleen by ELISA, Western blot and PCR. In mice infected with RH strain, circulating antigens and parasitemia were first detected from 2 days after infection, and Toxoplasma DNA were found in the blood, liver, brain and spleen from 3 days after infection. It was impossible to detect specific IgM and IgG antibodies to T. gondii, and any specific band was not found by Western blot. In mice infected with Beverley strain, circulating antigens were detected between day 10 and day 35. The Toxoplasma DNA was found in the blood and liver from day 15 until day 60, and in the brain from day 20. But Toxoplasma DNA in the spleen were mainly detected between day 10 and day 30. The IgM antibodies were first appeared on day 10 post-infection, and were noted obviously increased between day 15 and 25. The IgG antibodies were first detected on day 15, and showed progressively increased titers. The antibody binding bands were specific according to infection period. Sera from mice infected with Beverley strain reacted mainly with the antigen of 27.5-kDa and 32.5-kDa. In conclusion, mice infected with RH strain revealed Toxoplasma antigens strongly, but not antibodies. However, mice infected with Beverley strain revealed both the Toxoplasma antigens and antibodies. The present results showed that immune responses are different between avirulent and virulent T. gondii.     

Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae. It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Immune response in the hamster. VIII. Ig class differences in susceptibility to tolerance induction

A hemolytic plaque assay was used to quantitate the antibody response of the Syrian hamster after immunization with hen egg albumin (HEA). Whereas HEA in complete Freund's adjuvant (HEA-CF) induced a prolonged heterogeneous (IgM, IgG1 and IgG2) antibody response, the response to soluble HEA in saline (HEA-S) differed in that: 1)both the primary and secondary responses were restricted to the IgG1 class; 2)the IgG1 primary response was cyclical with PFC peaks on days 9 and 16; 3)although an anamnestic secondary response was demonstrated, no further augmentation was noted after tertiary and quaternary boosters; 4)the booster response was transient reaching a peak after 48 hr and declining to low levels within 7 days. Adoptive transfer of lymph node cells to irradiated recipients followed by challenge with HEA-CF revealed: 1)that HEA-S-treated donor cells were primed for an IgG1 response because anamnesis was seen 7 days after challenge, yet on day 21, IgG1 PFC were 20-fold less than that of controls; 2)recipients of HEA-S treated cells showed profound suppression of both IgM and IgG2 PFC on days 14 and 21. These studies indicate that soluble antigen induced in hamsters a state of complete tolerance of IgM and IgG2 classes whereas the anamnestic response of the IgG1 class remained intact.     

CD40 cross-linking inhibits specific antibody production by human B cells

Ligation of CD40 on B cells is a co-stimulatory signal for proliferation, antibody secretion, heavy chain switching and rescue from apoptosis after somatic mutation in the germinal centre. The importance of these manifold responses to CD40 activation for humoral immunity is exemplified by the inability of boys with X-linked hyper IgM syndrome to make IgG, IgE or IgA due to a mutation in in the gene coding for CD40 ligand (CD40L). In the present study, we have investigated the effect of CD40 ligation on specific antibody production by human B cells to influenza virus. The antibody response was T cell dependent and specific for the strain of influenza virus used as antigen. Addition of either CD40 mAb or recombinant trimeric CD40L profoundly inhibited specific antibody production. Antibody production by unseparated tonsillar mononuclear cells and by T-depleted B cells stimulated with antigen in the presence of T cell replacing factor were equally inhibited with CD40 antibody showing that the effect was due to ligation of CD40 on B cells rather than blocking of T cell help. The specific antibody detected in these experiments was mostly IgG with little or no IgM and was obtained from surface IgM B cells consistent with activation of a secondary (memory) response. Co-stimulation of tonsillar B cells with CD40 antibody and anti-IgG induced proliferation of IgG+ B cells. These results suggest that CD40 ligation can inhibit specific antibody responses and stimulate proliferation in the same IgG+ (memory) B cell subpopulation. Addition of CD40 antibody during the first 24-48 h of the response was required for inhibition, suggesting that the effect was on early B cell activation and/or proliferation required for antibody production. There was no correlation, however, between the ability of CD40 mAb to stimulate proliferation and inhibit antibody production. We suggest that early activation of CD40 in the specific antibody response inhibits the formation of plasma cells and promotes instead the generation of memory cells.     

Human lymphoblastoid cell line producing specific antibody against Rh-antigen D

A lymphoblastoid cell line producing specific anti-Rh antibody against D determinant was established by Epstein-Barr virus (EBV) infection. The lymphocytes were from an immunized male donor with high titre of anti-D antibody. The cells were preselected by rosetting them with Rh-positive erythrocytes before EBV infection. The cell line produced specific antibody of IgG class, namely IgG1 subclass, but it also produced nonspecific IgM and IgG. By rerosetting the cell line, the specific antibody-producing population could be enriched and the antibody titre increased in the culture supernatant to the level of immune sera.     

ISCOM vaccine against contagious bovine pleuropneumonia (CBPP). 1. Biochemical and immunological characterization

A better vaccine than the existing ones against contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) would improve the chances for eradication of CBPP. In such an effort, immunostimulating complexes (ISCOMS) have been prepared from the whole detergent-solubilized cells of MmmSC and characterized biochemically and immunologically. The most efficient detergent for solubilization of the mycoplasma was MEGA-10 which yielded a high recovery of proteins in the ISCOMS. The ISCOMS showed the typical cage-like structure by EM and sedimented as 19S by sucrose gradient centrifugation. The protein pattern of the ISCOMS, analyzed in SDS-PAGE, revealed a great number of bands distributed along the gel as high and low molecular weight polypeptides. The Western blot developed with a serum from a CBPP infected animal detected a reduced number of polypeptides. In samples from whole mycoplasma cells and in ISCOMS, lectin blots revealed more than 20 carbohydrate structures. The ISCOMS induced a strong primary antibody response in mice measured by ELISA and the boost resulted in a 6-fold increase of the serum antibody response. The IgG response was distributed into various IgG subclasses with high IgG1, IgG2a and IgG2b titres while the IgG3 response was low. In cattle the ISCOM vaccine induced strong primary and long lasting secondary antibody responses of similar magnitudes as those of naturally infected animals as recorded by ELISA which persisted more than a year. IgG response was equally distributed in IgG1 and IgG2 subclasses. Also a cell-mediated immune response measured by proliferation assay was induced by low dose of ISCOMS. In the growth inhibition test, sera from vaccinated cattle readily inhibited colony growth already after the first immunization.     

Effect of a monoclonal antibody against interleukin-4 on the induction of oral tolerance in mice

The present study was undertaken to investigate the effect of a monoclonal antibody against interleukin-4 on the induction of oral tolerance. Oral tolerance was induced by feeding mice with low and high doses (0.1, 1 and 10 mg) of hen egg lysozyme once a day for 5 days before immunization with the antigen. An anti-interleukin-4 monoclonal antibody was i.p. injected 30 min before each oral administration of hen egg lysozyme. The results showed that the oral administration of hen egg lysozyme suppressed immune responses to the antigen including delayed type hypersensitivity, production of both isotypes of immunoglobulin (Ig) G1 and IgG2a antibodies and proliferation of lymph node cells in a dose-dependent manner. The suppression of these responses by the oral antigen was associated with a marked reduction of interferon-gamma secretion and a moderate decrease in interleukin-4 production by lymphoid cells. The treatment with the anti-interleukin-4 monoclonal antibody blocked dose-dependently the suppression of the delayed type hypersensitivity response to hen egg lysozyme, anti-hen egg lysozyme IgG2a antibody production and interferon-gamma secretion. In contrast, the anti-interleukin-4 antibody facilitated the suppression of anti-hen egg lysozyme IgG1 antibody production and interleukin-4 secretion. Thus, the neutralization of interleukin-4 by anti-interleukin-4 antibodies appears to be effective in modulating the induction of oral tolerance.     

Effect of cytokine-specific antisense oligonucleotides on the immunoglobulin production by rat spleen cells in vitro

We examined in this study the regulation of immunoglobulin (IgM, IgG, IgE) production by spleen cells from N brasiliensis infected rats following addition of antisense oligodeoxynucleotides (ODNs). The ODNs were selected near the AUG initiation codon of mRNA specific for interleukin 4 (IL-4) or interleukin 2 (IL-2). Results show that addition of antisense to IL-4 inhibited IgE production, while the production of IgG and IgM increased. The use of sense IL-4 sequence did not affect immunoglobulin production. In contrast, the use of antisense IL-2 ODN induced an enhancement of IgE as well as of IgM and IgG responses. Both the Ig secretion in culture supernatants and the number of Ig secreting cells, as detected by an Elispot assay, were influenced by the presence of antisense IL-4 ODNs. These results clearly show the involvement of IL-2 and IL-4 in the regulation of isotype selection during antibody synthesis and that IL-2 and IL-4 do operate differently on IgE production. They also argue that antisense strategy represents a useful tool for the antibody regulation.     

Admission balance and outcomes of patients admitted for acute inpatient rehabilitation

BALB/c mice infested three times with nymphs or larvae of Ixodes ricinus ticks do not acquire resistance as assessed by evaluation of both tick attachment and the weight of engorged nymphs or larvae. Tick challenge causes a gradual increase in total IgE antibody production from the first to the third infestation. Anti-tick IgG antibodies are never detected. When the mice are treated with anti-interleukin-4 (anti-IL-4) or anti-interferon-gamma (anti-IFN-gamma) monoclonal antibodies (mAbs) 1 day before each infestation, they produce fewer or more IgE antibodies, respectively. No effect is observed on IgG antibodies. In IL-4-deficient mice, no IgE or IgG antibody is produced. However, these treatments and the use of IL-4-deficient mice have no negative effect on either tick attachment or the weight of engorged nymphs or larvae. Treatment with anti-IL-4 mAb and the use of IL-4-deficient mice inhibits and abolishes the switching of IgE, respectively, but these are apparently not sufficient to shift the response toward Th1 cells.     

Enzyme-linked immunosorbent assay for the detection of Cryptosporidium parvum IgG in the serum of cats

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium parvum IgG in the serum of cats. The ELISA was an indirect ELISA using soluble C. parvum oocyst antigens and a peroxidase-labeled anti-feline IgG secondary antibody. Sera from cats with Toxocara felis, Giardia spp., Aelurostrongylus abstrusus, Isospora felis, Isospora rivolta, Toxoplasma gondii, or Taenia spp. infections were assayed in specificity studies. Following optimization, the ELISA and fecal examination for oocysts were performed on samples from 170 client-owned or humane society source cats and 1 cat inoculated orally with C. parvum oocysts. Cryptosporidium parvum oocysts were detected in feces (4/170; 2.4%), and C. parvum IgG was detected in serum (26/170; 15.3%) from naturally exposed cats. The seroprevalence data suggest that some cats in the geographical area studied were exposed to C. parvum, but persistent oocyst shedding was less common. The ELISA is not useful for predicting oocyst shedding in individual cats.     

Local expression of tumor necrosis factor alpha and interleukin-2 correlates with protection against corneal scarring after ocular challenge of vaccinated mice with herpes simplex virus type 1

To correlate specific local immune responses with protection from corneal scarring, we examined immune cell infiltrates in the cornea after ocular challenge of vaccinated mice with herpes simplex virus type 1 (HSV-1). This is the first report to examine corneal infiltrates following ocular challenge of a vaccinated mouse rather than following infection of a naive mouse. Mice were vaccinated systemically with vaccines that following ocular challenge with HSV-1 resulted in (i) complete protection against corneal disease (KOS, an avirulent strain of HSV-1); (ii) partial protection, resulting in moderate corneal disease (baculovirus-expressed HSV-1 glycoprotein E [gE]); and (iii) no protection, resulting in severe corneal disease (mock vaccine). Infiltration into the cornea of CD4+ T cells, CD8+ T cells, macrophages, and cells containing various lymphokines was monitored on days 0, 1, 3, 7, and 10 postchallenge by immunocytochemistry of corneal sections. Prior to ocular challenge, no eye disease or corneal infiltrates were detected in any mice. KOS-vaccinated mice developed high HSV-1 neutralizing antibody titers (> 1:640) in serum. After ocular challenge, they were completely protected against death, developed no corneal disease, and had no detectable virus in their tear films at any time examined. In response to the ocular challenge, these mice developed high local levels of infiltrating CD4+ T cells and cells containing interleukin-2 (IL-2), IL-4, IL-6, or tumor necrosis factor alpha (TNF-alpha). In contrast, only low levels of infiltrating CD8+ T cells were found, and gamma interferon (IFN-gamma)-containing cells were not present until day 10. gE-vaccinated mice developed neutralizing antibody titers in serum almost as high as those of the KOS-vaccinated mice (> 1:320). After ocular challenge, they were also completely protected against death. However, the gE-vaccinated mice developed low levels of corneal disease and virus was detected in one-third of their eyes. Compared with KOS-vaccinated mice, the gE-vaccinated mice had a similar pattern of IFN-gamma, but a delay in the appearance of CD4+ T cells, CD8+ T cells, and IL-4-, IL-6-, and TNF-alpha-containing cells. In sharp contrast to those of the KOS-vaccinated mice, no cells containing IL-2 were detected in the eyes of gE-vaccinated mice at any time. Mock-vaccinated mice developed no detectable neutralizing antibody titer and were not protected from lethal HSV-1 challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

An Eikenella corrodens toxin detected by plaque toxin-neutralizing monoclonal antibodies

Bacterial plaque from the gingival region of teeth contains cytotoxic agents which lyse undifferentiated human HL60 cells. A small panel of monoclonal antibodies (MAbs) was found to abrogate much of this activity and to detect antigens in certain strains of Streptococcus mitis and Eikenella corrodens. The aim of this study was to determine whether these bacterial antigens might be involved in HL60 cells cytolysis. Saline extracts were obtained by homogenizing washed, stationary-phase cells in 65 mM NaCl with a tight-fitting Potter-Elvehjem homogenizer. The extracts of E. corrodens were toxic to HL60 cells, whereas similar extracts of S. mitis were nontoxic. Adding plaque toxin-neutralizing MAb 3hE5 blocked the toxic effect of E. corrodens extract S. mitis extracts contained a single, strongly reactive antigen of 140 kDa (s140K antigen) detected on Western blots (immunoblots) by three MAbs from the panel. Rabbit antibodies raised to this antigen excised from the gel (anti-s140K serum) detected larger antigens in addition to s140K. E. corrodens extracts contained a number of antigens detected by the MAbs. Immunoglobulin G (IgG) was purified from anti-s140K serum by passage through DE52 cellulose. A 100-fold excess (by weight) of the purified IgG to E. corrodens protein specifically cross-precipitated an 80-kDa antigen plus a nonantigenic 16-kDa protein, presumably attached noncovalently. The remaining supernatant fraction had no toxic activity. A similar ratio of control IgG (from nonimmunized rabbits) did not precipitate these proteins, and the supernatant fraction had the same activity as the extract not treated with IgG. The proteins of 80 and 16 kDa were also detected in the anti-s140K immunoprecipitate by rabbit IgG antibodies to E. corrodens whole cells. The 80-kDa antigen, alone or complexed with the 16-kDa protein, may be involved in mediating the toxic activity in E. corrodens and plaque extracts.     

Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv18 (overexpressed by ovarian carcinoma cells) as part of a phase I/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10(6)-10(9)) of bsAb-targeted T lymphocytes plus low-dose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10(9) targeted T lymphocytes, 2 mg soluble bsAb, and low-dose IL-2. Using enzyme-linked immunosorbent assays (ELISA), human anti-mouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (1) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human anti-mouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line Igrov-I by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.