Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor.

By the genetic selection of mouse cDNAs encoding secreted proteins, a B7-like cDNA clone termed mouse GL50 (mGL50) was isolated encoding a 322-aa polypeptide identical with B7h. Isolation of the human ortholog of this cDNA (hGL50) revealed a coding sequence of 309 aa residues with 42% sequence identity with mGL50. Northern analysis indicated GL50 to be present in many tissues including lymphoid, embryonic yolk sac, and fetal liver samples. Of the CD28, CTLA4, and ICOS fusion constructs tested, flow cytometric analysis demonstrated only mouse ICOS-IgG binding to mGL50 cell transfectants. Subsequent phenotyping demonstrated high levels of ICOS ligand staining on splenic CD19+ B cells and low levels on CD3+ T cells. These results indicate that GL50 is a specific ligand for the ICOS receptor and suggest that the GL50-ICOS interaction functions in lymphocyte costimulation.

Autocrine regulation of IL-12 receptor expression is independent of secondary IFN-gamma secretion and not restricted to T and NK cells.

The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12Rbeta1 and IL-12Rbeta2) that are expressed on NK cells and activated T cells. The selective loss of IL-12Rbeta2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12Rbeta2 expression can be prevented by treatment with IFN-gamma, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12Rbeta1 and beta2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN-gamma secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN-gamma secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN-gamma production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN-gamma secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation.

Role of IL-12 in the induction and potentiation of IFN-gamma in response to bacillus Calmette-Guérin.

Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been accepted as the most effective agent in clinical use against superficial bladder cancer, its mechanism of action remains incompletely understood. A kinetic analysis in assessing the potential role of cytokines from BCG-stimulated murine splenocytes showed that IL-12 expression preceded that of other cytokines. Experiments subtracting endogenous BCG-driven IL-12 using neutralizing Ab or augmenting its activity with supplemental rIL-12 revealed not only that IL-12 plays a dominant role in IFN-gamma induction but also that it is normally dose limiting. A striking increase in IFN-gamma production could be generated in both mouse and human immunocompetent cell culture by the addition of even a small amount of rIL-12. Moreover, this same synergistic effect could be replicated during in vivo administration of BCG plus rIL-12 into the mouse bladder and was observed in a patient receiving intravesical combination therapy. In costimulation cultures, this synergy appeared to partially rely on IL-18 and IL-2 and could be down-regulated by IL-10. This suggests that a dynamic interplay between Th1 and Th2 cytokines is responsible for net IFN-gamma production. The ability of supplemental exogenous IL-12 to strongly shift this balance toward Th1 provides an immunological basis for using it in conjunction with intravesical BCG for bladder cancer immunotherapy.

Systemic reactivation of otitis media with effusion in a rat model.

OBJECTIVE: This study addresses the interaction of bacterial antigens, specifically peptidoglycan-polysaccharide (PG-PS) and lipopolysaccharide (LPS), in the induction and reactivation of mucoid middle ear effusions. METHODS: Twenty-seven rats underwent eustachian tube obstruction before inoculation of the middle ear bulla with PG-PS. Three weeks later, after resolution of all middle ear effusions, 6 rats were randomly selected and euthanized as the first control group (control I). The remaining 21 animals were randomly assigned to 3 groups that received intravenous injections of Krebs Ringer (control II), PG-PS, and LPS, respectively. These rats were euthanized 2 days after intravenous injection. Middle ear mucin production and histologic changes were measured in all animals. RESULTS: The mean concentrations of mucin were 0.94 +/- 0.52 mg/mL, 0.41 +/- 0.87 mg/mL, 16.33 +/- 3.67 mg/mL, and 1.15 +/- 0.41 mg/mL in the control I, control II, PG-PS, and LPS groups, respectively. Thus the mean concentration of mucin in the middle ear lavage samples was significantly greater in rats that were injected intravenously with PG-PS than in rats in other groups (P < 0.05). Histologic analyses demonstrated a greater degree of goblet cell hyperplasia in the PG-PS group than in other groups. CONCLUSIONS: This is the first animal model of recurring otitis media with effusion in which a systemic injection of PG-PS was used to reactivate a middle ear effusion in rats previously primed with a transtympanic injection of PG-PS. This study suggests that after otitis media with effusion has resolved, it may be reactivated by the presence of bacterial antigens and/or cytokines in the systemic circulation.

Mucin production in the middle ear in response to lipopolysaccharides.

OBJECTIVE: This study examined the response of middle ear tissue to establish the lowest dose of lipopolysaccharide to induce mucin production in a rat otitis media model. METHODS: Twenty-six male Sprague-Dawley rats' eustachian tubes were obstructed before transtympanic inoculation of the bulla tympanica with 35 microL of Krebs Ringer or 1, 10, 100, or 1000 microgram/mL lipopolysaccharide. After 7 days the effusion and a lavage were collected for mucin ELISA measurement, and tissue was collected for histologic evaluation. RESULTS: Mucin secretion was significantly increased in the 100 microgram/mL 51.20 +/- 13.6 microgram/mL (SE) and 1000 microgram/mL 69.42 +/- 8.57 microgram/mL groups when compared with the Krebs Ringer control group 1.84 +/- 0.28 microgram/mL (P < 0.05). Histologic evaluation shows goblet cell metaplasia and hyperplasia in the middle ear epithelium in the 1000 and 100 microgram/mL groups. CONCLUSIONS: The histology and ELISA results suggest that a middle ear effusion is generated with a dose of lipopolysaccharide as low as 100 microgram/mL.

Immunoreactive macrophage colony-stimulating factor is increased in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits after recombinant human macrophage colony-stimulating factor therapy.

Infiltration of monocytes into arteries is an early event in the pathogenesis of atherosclerosis. This recruitment is interpreted as enhancing lesion development, but it could also be a host response limiting lipid accumulation. The ability of macrophages to limit cholesterol uptake, however, can be reduced by the impaired mobility and metabolic activity associated with foam cell development. As lesions enlarge, foam cells die and become the nidus for the necrotic core. Treatments to improve viability might improve foam cell function and promote regression. Macrophage colony-stimulating factor (M-CSF) is vital to monocyte/macrophage differentiation, proliferation, and activation. We found that foam cells of Watanabe heritable hyperlipidemic (WHHL) rabbits had faint staining for M-CSF. Treatment of rabbits with recombinant human M-CSF (rhM-CSF) increased M-CSF staining, which correlated with reduced cholesterol content of these foam cells.

Immunoregulation by interleukin-12 in MB49.1 tumor-bearing mice: cellular and cytokine-mediated effector mechanisms.

Administration of recombinant murine interleukin (rmIL)-12 to MB49.1 tumor-bearing mice results in dose-dependent regression of the primary tumor and the generation of protective antitumor immunity in the majority of animals. rmIL-12 administration is associated with a marked increase in lymph node cellularity that is predominantly due to the expansion of B220+ B cells as well as CD8+ T cells. Stimulation of lymph node cells from rmIL-12-treated, but not control tumor-bearing mice, with MB49.1 tumor cells in vitro was shown to enhance the secretion of interferon (IFN)-gamma. The magnitude of this in vitro response was dependent on the dose of rmIL-12 administered in vivo and mirrored the change in circulating serum IFN-gamma. Furthermore, at the height of the in vitro response to tumor stimulation, the addition of a neutralizing antibody to murine IL-12 suppressed IFN-gamma production, indicating a role for endogenous IL-12 in this antigen-specific cytokine response. Although studies in SCID mice confirmed that an appropriate T cell response was required for rmIL-12-mediated antitumor activity, in immunocompetent animals early tumor regression was not accompanied by cellular infiltration of the tumor. In contrast, a profound increase in tumor-associated inducible nitric oxide synthase (iNOS) was observed in mice receiving rmIL-12 which preceded T cell infiltration of the tumor which could be detected during the second week of IL-12 treatment. Direct tumor killing through the cytotoxic actions of NO via the iNOS pathway may serve as a way of generating tumor antigen which enables the host to mount a subsequent T cell response against the tumor.

Atypical zeta-protein kinase c displays a unique developmental expression pattern in rat brain.

The expression of atypical zeta-protein kinase C (PKC) was examined during prenatal and postnatal rat brain development. Immunoblot as well as transcript analysis revealed a dramatic increase in expression at 2-3 days post-birth, which declined thereafter and remained at levels observed in the adult brain. The expression of zeta-PKC precedes that of the other PKC isoforms in developing rat brain. Subcellular fractionation of pup and adult brain documented distribution between all three distinct fractions (A,B,C), including the low speed pellet composed of nuclei. In adult brain, the kinase was enriched in the A fraction of the sucrose gradient. Specific substrate proteins of zeta-PKC were characterized in each of the subcellular fractions from both pup and adult brain. Four predominant proteins pp76, pp60-doublet, pp54 and pp45 were identified as zeta-PKC endogenous substrates. All four proteins were phosphorylated on serine residues, while the pp60-doublet was also phosphorylated on tyrosine. The pp60-doublet was the most predominant substrate, specifically enriched in the A fraction of a sucrose gradient of adult brain and immunoprecipitated by monoclonal antibody to pp60c-src.

Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus.

Oligonucleotide probes designed on the basis of the N-terminal sequence of Clostridium perfringens beta-toxin were used to isolate the encoding gene (cpb). The nucleotide sequence of cpb was determined, and on the basis of DNA hybridization experiments it was shown that the gene is found only in type B and C strains of C. perfringens. The deduced amino acid sequence of the beta-toxin revealed homology with the alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus. The beta-toxin purified from C. perfringens appeared to exist in monomeric and multimeric forms. Recombinant beta-toxin, produced in Escherichia coli, appeared to be mainly in the multimeric form.

Comparison of the nucleotide sequence and development of a PCR test for the epsilon toxin gene of Clostridium perfringens type B and type D.

The sequence of the epsilon toxin gene of Clostridium perfringens type D was determined and compared with that of the previously reported type B sequence. It showed two nucleotide changes in the open reading frame, giving rise to one amino acid substitution. The promoter sequences were not homologous, and different putative -35 and -10 regions have been identified in each. The sequence information was used to develop PCR primers which were specific for the epsilon toxin gene. The utility of this system for identifying type B or D strains of C. perfringens was demonstrated.

Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli.

The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined. Some differences between this sequence and the previously published sequence (A. S. Bhown and A. F. S. A. Habeeb, Biochem. Biophys. Res. Commun. 78:889-896, 1977) were found. A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C. perfringens type B. The gene encoded a protein with a molecular weight of 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli sigma 70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001. Southern hybridization experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce epsilon-toxin.

Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.

Selective limbotomy.

A method is designed dividing the limbic system into sectors. The method was tested by analyzing 50 procedures carried out by the authors. Rostral limbotomies, performed either by open techniques (cingulectomy) or by stereotactic surgery, showed similar results, although recurrence was more frequent with stereotaxis. Comparison with other described procedures on the same areas revealed similar results. Analysis suggests that the interruption of the cingulum was responsible for the permanent behavior modification and not the interruption of intersecting anatomical systems.

Surgical treatment of 63 cases of conjoined nerve roots.

The operative results of 63 cases of lumbar disc disease with surgically confirmed conjoined nerve roots are reviewed. The first 55 patients were treated by standard hemilaminectomy and discectomy, with only 30% reporting a good result. Of the last eight patients treated by hemilaminectomy, pediculectomy, and discectomy, seven patients returned to work. Te rationale for and the technique of pediculectomy are discussed in detail. Clinical, radiological, and surgical clues indicating the presence of the conjoined nerve root anomaly are reviewed.

Intraspinal extradural sensory rhizotomy in patients with failure of lumbar disc surgery.

Questionnaires were sent to 60 patients who had undergone an intraspinal extradural sensory rhizotomy after failure of back surgery to assess the efficacy of the procedure. Questionnaires were returned from 47 patients. The operative results were uniformly poor in improving the level of activity of the patient. However, nearly 60% of the patients obtained relief of their pain. Section of only one root, either L-5 or S-1, relieved pain in 50% of the cases. Section of both roots, L-5 and S-1, appeared more effective, since 66% of these patients were relieved of their pain. The technique of performing an intraspinal extradural sensory rhizotomy is discussed in detail.

Assessment of the effects of cinglulate gyrus lesions by neuropsychological techniques.

Nineteen psychiatric patients undergoing bilateral cryogenic cingulate cortex lesions were extensively evaluated pre- and postoperatively with objective measures of intelligence, higher cortical functions, memory, and emotional status. Following surgery the patients as a group revealed no significant deterioration of functions; rather, they demonstrated improvement that could be interpreted as the result of decline in anxiety. Investigations of individual revealed that the overall test performance was improved in 13 and substantially unchanged in three, whereas three demonstrated some decline in performance. These results were discussed in terms of the characteristics of the changes across the various tests.

Pain suppression by peripheral nerve stimulation. Chronic effects of implanted devices.

This is a study of the long range effects of pain suppression obtained by electrical stimulation of peripheral nerves. These cases were followed during 12--46 months and evaluated personally and by questionnaires. Selection for surgery was done exclusively on the basis of the results of a preoperative peripheral nerve stimulation test. Of 37 cases observations, 18 were considered significantly relieved; that is, more than 50% of the intensity and/or duration of pain was consistently admitted. The results obtained in the acute preoperative trial could be reproduced indefinitely in some cases for as long as 46 months. Correlation of the results with the disease producing the pain revealed as benefitting for painful syndromes associated with peripheral nerve disorders, amputation, soft tissue injuries (nerves?), and some recurrent lumbar disc surgeries. Sciatic, ulnar and occipital nerve implantations were particularly rewarding. The best and worse results were analyzed. The complications appear to be largely preventable and of no serious consequences. Our analysis suggests that most failures take place within 2 years from implantation. Experience seems to be accumulating showing that a number of patients may receive sustained relief beyond this period.

Pain suppression by peripheral nerve stimulation. Part I. Observations with transcutaneous stimuli.

A systematic, strict appraisal was made of 100 patients, after preliminary clinical trials suggested that some patients with pain could be helped by peripheral nerve stimulation. Transcutaneous stimulation of different nerve trunks was done with a special electrical stimulation device with various selected electrical parameters. More than half of the patients experienced some relief; in many, this effect was obtained by stimulating nerves distant from the area of referred pain. Pain relief lasted for varying periods after stimulation. The maximum benefit was noticed after certain specific parameters were reached for each patient. A few patients had response decay, gain or worsening. Results differ to some degree from previous reports. The results seem encouraging for the treatment of certain forms of intractable pain.