Development of a receptor peptide antagonist to human gamma-interferon and characterization of its ligand-bound conformation using transferred nuclear Overhauser effect spectroscopy.

Polyclonal anti-idiotypic antibody raised to a synthetic discontinuous peptide derived from the human gamma-interferon (huIFN-gamma) sequence recognizes soluble human gamma-interferon receptor (Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994) J. Biol. Chem. 269, 358-363). We sought to use this reagent to identify a ligand-binding domain within IFN-gamma-receptor. To do this, the neutralizing anti-idiotypic antibody was used to probe overlapping linear peptide octamers of the extracellular domain of the huIFN-gamma receptor. A 22-amino-acid residue receptor segment 120-141 identified by the antibody was synthesized. CD and NMR analysis indicates that peptide 120-141 has no apparent secondary structure in water or in water containing 50% trifluoroethanol. The synthetic receptor peptide inhibited huIFN-gamma induced expression of HLA/DR antigen on Colo 205 cells with an approximate IC50 of 35 microM. Immobilized peptide specifically bound recombinant huIFN-gamma but did not bind human granulocyte-macrophage colony-stimulating factor on a microtiter plate in a direct binding enzyme-linked immunosorbent assay. The binding results are supported by two-dimensional transferred nuclear Overhauser effect (TRNOE) NMR data obtained on the peptide in the presence of recombinant huIFN-gamma. Characterization of the conformation of the bound peptide by TRNOE suggests that this peptide assumes a distinct conformation. Intramolecular interactions within the bound peptide were detected at two non-contiguous regions and at a third region comprising a beta-turn formed by the sequence DIRK. We believe that this represents the structure of the receptor within the ligand-binding domain.

Recognition by HLA-A2-restricted cytotoxic T lymphocytes of endogenously generated and exogenously provided synthetic peptide analogues of the influenza A virus matrix protein.

Experiments were carried out to determine whether complexes between MHC class I molecules and synthetic peptides are representative of those formed under more physiologically relevant conditions, with peptides derived intracellularly from processed antigens. Lysis of cells sensitized with exogenously provided and endogenously generated peptide analogues of the optimal nonameric peptide 58-66 (GILGFVFTL; derived from the influenza virus matrix protein) was compared. Endogenous loading was accomplished by expressing minigene DNA coding for alanine-substituted analogues of peptide 58-66 in HLA-A2-positive cells. Susceptibility to lysis by HLA-A2-restricted, peptide-specific cytotoxic lymphocytes was compared with lysis of cells sensitized with the same synthetic peptides. Although results were quite comparable, differences were observed. The endogenously presented analogues 58-66L60A, G61A, T65A, and L66A were recognized more efficiently than the corresponding exogenously presented analogues. This difference in recognition was most striking for peptide 58-66G61A. These results indicate the need for caution in using synthetic peptides in defining peptide binding motifs. Additional experiments with endogenously expressed analogues of 58-66 with substitutions other than alanine were carried out to define the interaction between this peptide and HLA-A2. Results are compatible with the interpretation that residues 58, 59, and 60 interact with pockets A, B, and D, respectively, in the HLA-A2 binding groove and that these interactions contribute to peptide binding.

Presentation of endogenous peptides to MHC class I-restricted cytotoxic T lymphocytes in transport deletion mutant T2 cells.

The ability of minigene-encoded viral peptide epitopes to be presented by class I molecules in the absence of MHC-encoded transporters has been evaluated in mutant T2 cells. These cells have a large deletion in the class II MHC region that includes the known transporter protein for antigenic peptides and proteasome genes and they are defective in presenting viral epitopes to CTL. T2 cells that express minigenes encoding the influenza virus matrix peptide 58-66 (GILGFVFTL) and two HTLV 1 Tax peptides 11-19 (LLFGYPVYV) and 12-19 were lysed by HLA-A2-restricted peptide-specific CTL. Minigene expression of a HLA-A2-restricted HIV reverse transcriptase peptide 476-484 (ILKEPVHGV) with three charged residues sensitized T2 cells poorly for lysis by HIV-specific CTL unless the peptide was preceded by an endoplasmic reticulum translocation signal sequence. Expression of an influenza virus nucleoprotein peptide 383-391 (SRYWAIRTR) with three charged arginine residues did sensitize HLA-B27+ T2 cells for lysis by peptide-specific CTL. These and other results with endogenously expressed peptide analogs in which hydrophobic and charged amino acids were interchanged demonstrate that antigenic peptides can be translocated from the cytoplasm into the class I Ag presentation pathway independent of MHC-encoded transporters; and that peptide hydrophobicity appears not to be a major determinant in selecting peptides for this alternate pathway.

Identification of functionally distinct domains of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40-77, 78-94, or 110-127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

Treatment of choroid plexus papillomas in children: a brief analysis of twenty years' experience.

This is a brief report of 17 histologically verified choroid plexus papillomas (CPPs). The radiological evaluation and surgical treatment are outlined. The results indicate that the surgical approach was not optimal. Radiation therapy decreasing the vascularity of tissue-proven CPPs and therefore aiding the operative removal is illustrated. The limited experience with postoperative irradiation is presented. The computed tomography of this lesion is discussed.

Multiple nevoid basal-cell carcinoma syndrome (Gorlin's syndrome): possible confusion with metastatic medulloblastoma. Case report.

The authors report a diagnostic dilemma involving a child who, 8 years previously, had total excision of a medulloblastoma. On x-ray studies, lytic lesions of the skull were seen. The differential diagnosis and some of the clinical and pathological aspects of the nevoid basal-cell carcinoma syndrome versus metastases are discussed.

Isolated fourth ventricle as a complication of ventricular shunting. Report of three cases.

Signs of cerebellar dysfunction combined with signs suggestive of shunt malfunction developed in three children with obstructive hydrocephalus. Shunt function was normal. In all cases, the cerebellar signs persisted and computerized tomography scans revealed enlargement of the fourth ventricle. Shunting of the fourth ventricle returned the patients to normal function.

Cervical myelopathy due to spondylosis. Case report.

The authors report the case of a patient with cervical myelopathy who was examined at autopsy 2 years after a second anterior cervical fusion by Cloward's technique. The clinical course and pre- and postoperative myelograms are presented. Theories as to the etiology of myelopathy are discussed. This case demonstrates chronic changes that seem to implicate a vascular theory but not the specific vessel or vessels. The mechanism of improvement following the Cloward procedure is not explained by the pathological slides.

The handling of animal wastes.

Most farm problems with animal wastes occur in modern intensive livestock enterprises where manure is handled as a slurry. It is not practical to treat slurry in the same way as domestic sewage: it should be used on land as a source of plant nutrients. On most farms, this can be done only at certain times of the year and so slurry has to be stored. Storage gives rise to problems of mixing, handling, application, pollution, smell and pathogen survival which can often be solved by separating slurry with special machinery into solid and liquid fractions. Where odour is a serious problem, however, some form of limited aeration will usually provide the best solution. For intensive pig units on limited land close to houses, the NIAE has evolved a new system of slurry treatment which can convert all the slurry from a fattening piggery into inoffensive solids. When incorporated into a piggery for 500 pigs being planned by the Ministry of Agriculture, Fisheries and Food, the system should also reduce smell substantially both inside and outside the building.