Mixture Of Monoclonal Antibodies Research Articles

Effect of Saquinavir on proliferation and telomerase activity of human peripheral blood mononuclear cells

The present study describes the effect of Saquinavir on proliferation, interferon-γ production and telomerase activity of non-stimulated, or activated non-adherent mononuclear cells (NAMNC), obtained from peripheral blood of healthy donors. Fresh NAMNC, non-stimulated or activated in vitro with PHA or with a mixture of monoclonal antibodies against CD3 and against CD28 membrane antigens (in order to obtain prevalent T cell responses), were exposed to Saquinavir before or at the time of mitogenic stimulation. Control and treated cells were tested for DNA synthesis ( 3H-thymidine incorporation), interferon-γ production and telomerase activity (TRAP assay). The results indicate that Saquinavir is able to increase proliferation and interferon-γ release in PHA-stimulated NAMNC, and telomerase activity either in non-stimulated and in PHA or antibody-activated cells. These results suggest that the activity against HIV infection afforded by Saquinavir, could be corroborated by its effects on the host. These include its adjuvant activity on mitogen-induced responses of lymphocytes, and its possible antagonistic effects against lymphoid cell senescence, through telomerase activation.

Enzyme-linked immunosorbent assay (ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening.

We have developed a solid-phase ELISA for the specific and sensitive detection of apoptotic cells. This method is based on the ability of a monoclonal antibody (MAb) against single-stranded DNA (ssDNA) to specifically identify apoptotic cells. The assay involves binding of cells to 96-well microtiter plates, treatment of the attached cells with formamide to denature DNA in apoptotic cells and one-step staining of the denatured DNA with a mixture of anti-ssDNA MAb and peroxidase-conjugated anti-mouse IgM. A near linear increase in signal was seen as the number of apoptotic cells increased from 500 to 5000. Untreated and necrotic cells or cells with single-stranded DNA breaks induced by H 2O 2 did not produce signal above the background level. In leukemic cell cultures grown, treated with ID 50 concentration of etoposide, stained and analyzed in the same 96-well assay plate, intense ELISA signal was detected. The ratio of absorbance values from drug resistant and drug-sensitive cell lines treated with etoposide was in agreement with the degree of resistance determined by growth inhibition assays. These data show that this ELISA has sufficient sensitivity for use in drug screening protocols. In breast cancer cell cultures treated with cisplatin, ELISA absorbance increased only after treatment with drug concentrations 10-fold higher than concentrations inducing 95% growth inhibition. In cultures treated with staurosporine, there was a near linear relation between the ELISA absorbance values and cytotoxicity in the range of 15–92% growth inhibition. The absence of apoptotic signal in breast cancer cells treated with cytotoxic concentrations of cisplatin indicated that this drug kills cells by non-apoptotic mechanisms, whereas apoptosis was the dominant mechanism of cell death caused by staurosporine. The formamide-MAb apoptosis ELISA described here may provide a basis for high-throughput screening of drugs based on their ability to induce or suppress apoptosis.

An immunoassay for small analytes with theoretical detection limits.

A flow-based immunoassay that uses microspheres as the solid phase accomplished the theoretical limit of detectability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was briefly exposed to a bead pack containing immobilized estriol in a flow cell. A small portion of free antibody was separated rapidly from the mixture by binding it to immobilized hormone, but the antibody-hormone complex was kinetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labeling the separated antibodies on the beads with a Cy5-conjugated antispecies secondary antibody. By labeling after the separation step, perturbing the liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumulating free antibody onto the beads prior to quantification and, thus, offered ideal conditions to achieve theoretical limits of detectability. The optimum achievable dynamic range of this immunoassay was 4-300 pM. Because the proportion of free anti-estriol antibody in the mixture was controlled by the Kd of the antibody-estriol interaction, when the concentration of the antibody was below the Kd, the smallest detectable estriol concentration approached the theoretical limit of detectability achievable with this antibody.

GLYCYRRHIZIN RESTORES THE IMPAIRED IL-12 PRODUCTION IN THERMALLY INJURED MICE

Mice 6 days after thermal injury (TI-mice) did not respond to lipopolysaccharide (LPS) stimulation for production of serum interleukin 12 (IL-12; 2h after LPS stimulation, <20pg/ml in TI-mice; 1091±162pg/ml in normal mice). However, 2h after LPS stimulation, 1456±118pg/ml of IL-12 were demonstrated in sera of TI-mice previously treated with a 10mg/kg i.p. dose of glycyrrhizin (GR). IL-12 was not induced by LPS in sera of normal mice inoculated with burn-associated type 2 T cells (IL-4/IL-10-producing CD8+CD11b+TCRγ/δ+T cells isolated from spleens of TI-mice). However, IL-12 production was induced by LPS in sera of these mice previously treated with GR or a mixture of monoclonal antibodies (mAbs) for type 2 cytokines. Also, IL-12 production was induced by LPS in TI-mice inoculated with CD4+T cells from spleens of GR-treated normal mice (GR-CD4+T cells, 5×106cells/mouse). Since GR-CD4+T cells have been shown to be antagonistic cells against production of type 2 cytokines by burn-associated type 2 T cells, these results indicate that IL-12 unresponsiveness shown in TI-mice is recovered by GR through the regulation of burn-associated type 2 T cell responses.

Saquinavir Up-Regulates Telomerase Activity in Lymphocytes Activated with Monoclonal Antibodies Against CD3/CD28

The present study describes the effect of the HIV protease inhibitor saquinavir on telomerase activity and interferon-γ (IFN-γ) production of non-adherent mononuclear cells (NA-MNC). Cells obtained from peripheral blood of healthy donors were exposed in vitro to a mixture of monoclonal antibodies against CD3 and CD28 membrane antigens in order to activate prevalently T cell subsets. Treatment with saquinavir was performed at the time of cell stimulation. Thereafter, NA-MNC were tested for telomerase activity (TRAP assay) and interferon-γ production up to 7 days later. The results show that saquinavir up-regulates telomerase activity and IFN-γ release in activated NA-MNC. These observations suggest that the anti-HIV effects of saquinavir could be accompanied by other immunopharmacological properties, influencing some aspects of the functional activity of immunocompetent cells. These include possible antagonistic effects against lymphocyte senescence, through telomerase activation, and a potentiating activity on the production of IFN-γ following T cell activation.

Influence of type 2 T cell responses on the severity of encephalitis associated with influenza virus infection

The role of type 2 T cell responses on the severity of post-infectious encephalitis was investigated in a mouse model of influenza virus infection. When mice were infected intracerebrally with 3.0 LD(50) of A/NWS33 strain of influenza virus, they all showed clinical signs of encephalitis, and 90% of them died within 10 days of the infection. However, the post-infectious encephalitis was not demonstrated in mice exposed to 0.5 LD50 of the same virus. The mortality rates of mice infected with 0.5 LD(50) of the virus were increased to levels observed in mice exposed to 3.0 LD(50) of influenza virus infection, after the administration of a mixture of interleukin (IL)-4 and IL-10 (2 ng/mouse each; immediately, 1 and 2 days after the infection). In contrast, mortality rates of mice exposed to 3.0 LD(50) of influenza virus were substantially decreased when these mice were treated with a mixture of monoclonal antibodies directed against IL-4 and IL-10. A predominance of type 2 T cell responses was demonstrated in splenic T cells of mice infected with 3.0 LD(50) of influenza virus, although these responses were minimal in mice infected with 0.5 LD(50) of the virus. After the treatment with the mixture of type 2 cytokines, an increase in the type 2 T cell responses in mice exposed to 0.5 LD(50) of the virus was shown. These results indicate that type 2 T cell responses associated with the viral infection play an important role in the severity of post-infectious encephalitis induced in mice by the intracerebral infection of influenza A virus.

Evidence that collapsin response mediator protein-2 is involved in the dynamics of microtubules.

Collapsin response mediator protein-2 (CRMP-2) is a member of the CRMP/TOAD/Ulip/DRP family of cytosolic phosphoproteins involved in neuronal differentiation and axonal guidance. CRMP-2 mediates the intracellular response to collapsin 1/semaphorin 3A, a repulsive extracellular guidance cue for axonal outgrowth. The mutation of UNC-33, a Caenorhabditis elegans homolog of CRMP-2, results in abnormality of microtubules in neurites, but the mechanism of CRMP-2 action remains to be clarified. Here, we report that overexpression of human CRMP-2 in Neuro2a cells, a mouse neuroblastoma cell line, results in blebbing of the cytoplasm. Furthermore, some cells exhibited intranuclear inclusions, which were labeled with antibodies to CRMP-2 and tubulin. CRMP-2 was found to be associated with microtubule bundles in the spindles at the metaphase and in the midbodies at the late telophase in mitotic cells. Thus, it is most likely that failure of complete disassembly of the spindle microtubules during mitosis is responsible for the formation of these intranuclear inclusions. We suggest that CRMP-2 functions by regulating the dynamics of microtubules.

Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies

Outer membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS), the main surface antigens of Brucella ovis, display surface-exposed epitopes. Mixtures of monoclonal antibodies (mAbs) to both antigens were previously shown to protect mice against a B. ovis challenge. To further identify the antigens involved, seven mAbs against Brucella OMPs (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1) and three to R-LPS were tested for protection either individually or in combinations. Significant reduction in spleen infection in challenged mice, relative to controls, was used as the protection criteri. Controls included nonimmunized mice and mice given an irrelevant, anti-O-polysaccharide (OPS), mAb. For comparison, a group received a mouse serum containing antibodies to both OMPs and R-LPS; this serum was prepared by immunization with a B. ovis hot-saline extract which, as described previously, induces protective immunity in mice and rams. Significant protection was observed with both mAbs to OMPs and R-LPS. mAbs to Omp16, Omp19 and Omp31 afforded the highest protection and prevented the development of splenomegaly. The protective effect of mAb to Omp31 was not interfered with by nonprotective mAbs in different mixtures. The data presented confirm the protective role of antibodies to OMPs and R-LPS against B. ovis, and identify several OMPs, especially Omp31, which are promising candidates for a subunit vaccine against ram epididymitis.

Glycoprotein Ib Is Homogeneously Distributed on External and Internal Membranes of Resting Platelets

Recent ultrastructural studies have suggested that Glycoprotein Ib (GPIb) has a different distribution on external (surface) versus internal (open canalicular system) membranes in resting discoid platelets. The differential distribution proposed for GPIb differs from that reported for the fibrinogen receptor, GPIIb-IIIa, and could have profound physiological significance when platelets are activated by surfaces. The present study explored the distribution of GPIb on external and internal membranes of resting platelets. Immunogold cytochemical techniques were applied to ultrathin cryosections of washed platelets. Polyclonal antibodies or mixtures of monoclonal antibodies (AP1 and 6D1) were used for labeling. To avoid the technical problem posed by limited accessibility of antigens located in very narrow portions of the open canalicular system (OCS) to antibodies, the same methods were applied to patients with giant platelets syndromes. The OCS of normal resting platelets was also dilated by exposure of platelets to hypertonic conditions or to cytochalasin-B, an agent that prevents assembly of actin, and, reportedly, movement of GPIb. Morphometric analysis revealed that rates of labeling on internal versus external membranes of giant platelets does not differ significantly (0.93 +/- 0.20), provided the OCS is sufficiently dilated. Platelets exposed to cytochalasin B (1.01 +/- 0.31) or to hypertonic conditions (0.96 +/- 0.20) revealed similar ratios for immunogold particles on external and internal membranes. Results of our study indicate that membranes of the exposed surface and lining OCS channels of resting platelets are continuous, identical structures and GPIb is homogeneously distributed on external and internal membranes.

Course and composition of the nerves that supply the mandibular teeth of the rat.

The rodent dentition has become an important model for investigations of interactions between dental tissues and peripheral neurons. Although experimental nerve injury has been widely used for such studies, there is uncertainty about the courses of nerve fibers supplying the mandibular teeth. In order to clarify this, we used a mixture of monoclonal antibodies against neurofilament proteins to enhance demonstration of nerve fibers so that small nerves could be readily traced in serial frozen sections of mandibles of Sprague Dawley rats ranging in age from embryonic day (E) 18 to postnatal day (P) 90. The 1st molar and anterior portion of the 2nd molar were innervated by small nerves that emerged as distinct branches of the IAN trunk at or near the mandibular foramen. In contrast, the nerve supply to the 3rd molar and posterior part of the 2nd molar was a branch of the lingual nerve that bypassed the mandibular canal altogether. The IAN trunk split into the mental nerve and a large branch to the incisor about 2 mm anterior to the mandibular foramen. Thick branches of the incisor nerve descended into the incisor socket to form a dense plexus of nerve fiber bundles extending along the length of the incisor periodontium. The sparse pulpal innervation of the incisor was provided by a few thin fascicles that emerged from the caudal portion of the periodontal plexus to enter the incisor apex. The dental branches of the IAN and lingual nerve seen in the adult were well established and readily identifiable at age E18 even though their targets were limited to the follicles of the developing teeth. These studies show that the trigeminal branches that supply the mandibular teeth can be identified at a wide range of ages as distinct nerves at a considerable distance proximal to their targets. This detailed information on the courses taken by the dental nerves can provide an anatomical basis for increased precision in characterization and perturbation of neural pathways from the molars and incisor.

Simultaneous Determination of α-Fetoprotein and Carcinoembryonic Antigen in Human Serum by Time-Resolved Fluoroimmunoassay

A novel simultaneous measurement method for α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti-AFP antibody labeled with BHHCT–Eu3+ and biotinylated anti-CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti-AFP antibody at 615 nm. Then a BHHCT–Sm3+-labeled streptavidin–bovine serum albumin conjugate (SA–BSA) was added to react with the biotinylated anti-CEA antibody. After the reaction, the unreacted SA–BSA was washed out, and a 0.1 M NaOH solution containing 1.0 × 10−5 M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA–BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA–BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90–110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.

Development of a highly sensitive enzyme immunoassay for human calcitonin using solid phase coupled with multiple antibodies.

We have developed a highly sensitive chemiluminescent enzyme immunoassay for human calcitonin using three different mouse monoclonal antibodies that recognize the N-terminal, C-terminal and central portions of a human calcitonin molecule. The assay signal in a two-step sandwich enzyme immunoassay for human calcitonin using a solid phase coupled with a mixture of monoclonal antibodies. CT08 and OCT1, was 3.7-fold higher than when using either or both solid phases coupled with CT08 or OCT1, respectively. This enhancement is the result of improved avidity of immobilized antibodies and greater stability of the complex of immobilized antibodies and calcitonin in the first reaction, which resulted in greater reactivity of the immunocomplex with alkaline phosphatase-conjugate in the second reaction. The present assay showed a linear response up to 2.5 micrograms/L of human calcitonin and a high specificity for human calcitonin, but not for rat calcitonin, human calcitonin gene-related peptide and rat calcitonin gene-related peptide. The detection limit of human calcitonin was estimated to be 0.29 ng/L at zero (assay blank) + 3 SD. Interbatch coefficients of variation ranged from 2.2-26.7%.

Membrane active lipids in remnant lipoproteins cause impairment of endothelium-dependent vasorelaxation.

We have recently found that remnant lipoproteins (RLPs) and their lipid fractions impair endothelium-dependent vasorelaxation (EDR). This study was aimed at clarifying mechanisms responsible for RLP-induced endothelial dysfunction in isolated rabbit aortas. RLPs were isolated from plasma in hyperlipidemic subjects by use of the immunoaffinity gel mixture of anti-ApoA1 and anti-ApoB100 monoclonal antibodies and ultracentrifugation. Organ chamber experiments showed that EDR impairment was restored by addition of reduced glutathione (GSH) or N-acetylcysteine, antioxidants, into the incubation buffer containing isolated rabbit aortas and RLPs (0.75 mg of triglyceride/mL). Furthermore, the incubation of isolated human red blood cells (RBCs) with RLP and its lipids converted the normal shape of RBCs to echinocytes, but coincubation with antioxidants suppressed the RLP-induced RBC transformation, suggesting that they exerted oxidative damage on RBC surface membranes. Studies with HPLC and the postcolumn chemiluminescence method showed that RLPs contain a substantial amount of phosphatidylcholine hydroperoxides. Peroxidized phosphatidylcholine also impaired EDR and had echinocytogenic action, both of which were suppressed by N-acetylcysteine. RLPs isolated from the plasma of patients under treatment with alpha-tocopherol, an antioxidant, had a lower level of phosphatidylcholine hydroperoxides (15% of the amount in nontreated patients), which was associated with a lack of the inhibitory action on EDR and with lesser effect on RBC transformation. Oxidative damage caused by lipid components in RLPs, especially peroxidized phospholipids, deteriorates cell surface membrane and may be at least partly responsible for RLP-induced impairment of EDR.

Biological properties of T6S cells, a clone of burn-associated type 2 T cells

A clone of type 2 T cells (T 6 S cell) was established from burn-associated CD 8+ CD 11 b+ TCR gamma/delta + type 2 T cells, and their biological properties were examined. After stimulation by anti-CD 3 monoclonal antibody (mAb), T 6 S cells produced IL-4 and IL-10 into their culture fluids. However, IFN-gamma and IL-2 were not demonstrated in the culture fluids of T 6 S cells stimulated with the mAb. The susceptibility of thermally injured mice to infection with herpes simplex virus type 1 (HSV-1) or Candida albicans (C. albicans) has been shown to be approximately 50 (C. albicans) to 100 (HSV-1) times greater than that of normal mice. As compared with the resistance of thermally injured mice to these pathogens, mice inoculated with 1 x 10(6) cells/mouse of T 6 S cells (designated as T 6 S-mice) showed the same susceptibility to the infections. When T 6 S-mice treated with a mixture of monoclonal antibodies (mAbs) directed against IL-4 and IL-10 were infected with a lethal dose of HSV-1 or C. albicans, their resistance to these pathogens was restored to levels found in normal mice. From the experiments using T 6 S-mice, it is suggest that type 2 T cells or their cytokine products (IL-4 and IL-10) may play an important role on the impaired resistance of thermally injured mice to certain opportunistic pathogens. T 6 S cell may be a useful tool to study opportunistic infections in hosts bearing type 2 T cells.

Enhanced neutralization of human respiratory syncytial virus by mixtures of monoclonal antibodies to the attachment (G) glycoprotein.

Neutralization of human respiratory syncytial virus (HRSV) by monoclonal antibodies specific for the G protein was evaluated in a microneutralization test. Only certain antibodies showed some degree of neutralization, reflected in a reduction of virus titre (10-20 times maximum), compared with negative controls. In contrast, a pool of antibodies that recognized conserved, group-specific and strain-specific epitopes showed a significant increase in virus neutralization (up to 500-1000 times). By testing binary, tertiary and quaternary combinations, four antibodies were identified which showed maximal effect in the neutralization test. These findings are discussed in terms of the location of antibody binding sites in the G protein primary structure and their relevance for HRSV neutralization and immunobiology.

Symptoms, aetiology and serological analysis of sweet potato virus disease in Uganda

Sweet potato virus disease (SPVD) is the name used to describe a range of severe symptoms in different cultivars of sweet potato, comprising overall plant stunting combined with leaf narrowing and distortion, and chlorosis, mosaic or vein‐clearing. Affected plants of various cultivars were collected from several regions of Uganda. All samples contained the aphid‐borne sweet potato feathery mottle potyvirus (SPFMV) and almost all contained the whitefly‐borne sweet potato chlorotic stunt closterovirus (SPCSV). SPCSV was detected by a mix of monoclonal antibodies (MAb) previously shown to react only to a Kenyan isolate of SPCSV, but not by a mixture of MAb that detected SPCSV isolates from Nigeria and other countries. Sweet potato chlorotic fleck virus (SPCFV) and sweet potato mild mottle ipomovirus (SPMMV) were seldom detected in SPVD‐affected plants, while sweet potato latent virus (SPLV) was never detected. Isolates of SPFMV and SPCSV obtained by insect transmissions together induced typical symptoms of SPVD when graft‐inoculated to virus‐free sweet potato. SPCSV alone caused stunting and either purpling or yellowing of middle and lower leaves when graft‐inoculated to virus‐free plants of two cultivars. Similarly diseased naturally inoculated field plants were shown consistently to contain SPCSV. Both this disease and SPVD spread rapidly in a sweet potato crop.

Remnant-like very-low-density lipoprotein isolated from hypertriglyceridemic patients by immunoaffinity chromatography suppressed 3-hydroxy-3-methylglutaryl coenzyme A activity of cultured human skin fibroblasts

It has been previously reported that VLDL unbound to monoclonal antibody against apoB-100 was rich in apoE, thus resembling remnant particles (J Lipid Res, 1993:33; 369–380). In the current study, we have further analyzed the unbound VLDL fraction in plasma from hypertriglyceridemic patients using a mixture of monoclonal antibodies against apoB100 and apoA-1. The unbound VLDL isolated from the plasma of hypertriglyceridemic patients was found to be rich in apoE, apoB-48, and triglyceride compared with the bound VLDL. Furthermore, these unbound VLDL, but not bound VLDL, significantly suppressed HMG CoA reductase activity of cultured human skin fibroblasts ( − 20 to − 25%, P = 0.0022). The degree of suppression is significantly correlated with the apoE content of unbound VLDL ( r = − 0.769, P < 0.05). Unbound VLDL failed to suppress the activity of HMG CoA reductase of LDL receptor negative fibroblasts. These observations indicate a potential atherogenicity of remnant-like unbound VLDL by delivering more cholesterol through the LDL receptor dependent pathway with apoE as a ligand. In conclusion, this new immunoaffinity chromatography system is a useful method for directly quantifying atherogenic remnants in plasma.

Cytomegalovirus Antigenemia in Acquired Immunodeficiency Syndrome Patients With Untreated Cytomegalovirus Retinitis

To determine the frequency of cytomegalovirus (CMV) viremia in patients with acquired immunodeficiency syndrome (AIDS) and untreated CMV retinitis using conventional cell culture isolation and the sensitive CMV antigenemia assay. We examined 24 AIDS patients with ophthalmologic diagnosis of untreated CMV retinitis and 24 AIDS patients without present or past retinitis (control patients) from three medical centers between September 1992 and March 1994. Cytomegalovirus antigenemia was detected by an indirect peroxidase staining in 300,000 cytocentrifuged neutrophils, using a mixture of murine monoclonal antibodies directed against the pp65 lower matrix protein of CMV. Positive antigenemia was demonstrated in eight (33.3%) of the 24 retinitis patients and in none of the 24 control patients (P < .001). Only two of the eight antigenemia-positive patients had a concurrent positive CMV isolation from blood leukocytes by conventional cell culture assay. These results emphasize the risk of extraocular disease in AIDS patients with CMV retinitis because the virus is often present in peripheral blood leukocytes. The CMV antigenemia assay may be a simple and rapid means of identifying those patients with unilateral retinitis at highest risk of developing CMV retinitis of the fellow eye or of visceral CMV disease if intravitreal injections or implants are used as sole treatment for CMV retinitis.

Interleukin-12 protects thermally injured mice from herpes simplex virus type 1 infection.

Severe burn injury is associated with increased susceptibility to severe herpesvirus infections. Type 2 cytokines [interleukin (IL)-4 and IL-10] released from burn-associated CD8+ type 2 T cells (BA-type 2 T cells) have been shown to play a role in the increased susceptibility of thermally injured mice (TI-mice) to herpes simplex virus type 1 (HSV-1) infection. Because IL-12 has been shown to inhibit the generation of type 2 T cells, murine rIL-12 was injected into TI-mice exposed to HSV-1 to determine whether IL-12 could influence HSV-1 infections in individuals bearing type 2 T cells. rIL-12 improved the resistance of TI-mice or mice inoculated with T6S cells (a BA-type 2 T cell clone) against HSV-1 infection. Type 2 cytokines were detected in sera of TI-mice or mice inoculated with T6S cells (T6S-mice). However, treatment of TI-mice or T6S-mice with rIL-12 inhibited type 2 cytokine production in the sera of these mice. All TI-mice exposed to a lethal dose of HSV-1 survived when they were treated with a mixture of monoclonal antibodies (mAbs) against type 2 cytokines. Staphylococcal enterotoxin A [an interferon-gamma (IFN-gamma) inducer] stimulated serum IFN-gamma production in TI-mice and T6S-mice treated with rIL-12, whereas no IFN-gamma was produced in mice treated with saline. These results suggest that IL-12 has the potential to protect TI-mice infected with a lethal dose of HSV-1 via a shift to type 1 T cell responses from type 2 T cell responses.

Altered patterns of migration of cytokine‐producing T lymphocytes in skin‐grafted naive or immune mice following in vivo administration of anti‐VCAM‐1 or ‐ICAM‐1

Naive or preimmunized (to B10.BR or BALB.k) C3H/HeJ mice received skin grafts from multiple minor histoincompatible B10.BR or BALB.k mice following antigen-specific portal venous (p.v.) pretransplant transfusion, a protocol known to produce prolongation of graft survival in naive animals. In addition, groups of mice received intravenous (i.v.) infusion following transplantation with a mixture of monoclonal antibodies (mAb) to vascular adhesion molecule-1L: very late activation antigen-4 (VCAM-1:VLA-4) or intracellular adhesion molecule-1:lymphocyte function-associated antigen-3 (ICAM-1:LFA-1). Cells were harvested from different tissues of the grafted mice at various times post grafting. RNA was extracted and analysed, using polymerase chain reaction, for expression of different cytokines potentially involved in the regulation of graft rejection [interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumour necrosis factor-alpha, interferon-gamma and transforming growth factor-beta]. In addition, using limiting dilution analysis, we investigated the frequency of allo-specific and third-party reactive cells producing IL-2 and IL-4 in vitro in different tissues of grafted mice following these treatments. The mAb treatment protocol which produced optimum increases in graft survival in naive versus immune mice was different, with anti-LFA-1:ICAM-1 superior for naive mice compared with anti-VLA-4:VCAM-1, and vice versa for immune animals. However, in each case, increased survival was associated with increases local to the graft in the frequency of occurrence of antigen-specific type-2 cytokine-producing cells.