Reversal of experimental autoimmune encephalomyelitis by a soluble peptide variant of a myelin basic protein epitope: T cell receptor antagonism and reduction of interferon gamma and tumor necrosis factor alpha production.

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in lesions of multiple sclerosis (MS) and in experimental allergic encephalomyelitis (EAE). T cells found in EAE lesions bear the same amino acids in the third complementary determining region of the T cell receptor (TCR) as those found in MS lesions. We analyzed the trimolecular interactions between MBP p87-99, class II major histocompatibility complex (MHC), and TCR, and designed soluble inhibitors for therapy. F, N, I, and V at positions 90, 92, 93, and 94 interact with MHC, whereas K, T, and P at positions 91, 95, and 96 interact with TCR. The peptides, p87-99[95T > A] and p87-99[96P > A] could compete more effectively with p87-99 for binding to MHC and could antagonize the in vitro response to T cells to p87-99 more effectively than p87-99[91K > A]. However, only p87-99[91K > A] prevented and reversed EAE, indicating that the extent of MHC or TCR competition does not predict success in treating EAE. To elucidate the mechanism of inhibition of EAE, draining lymph node cells from rats immunized with the native peptide alone or together with each of the three TCR antagonists were challenged in vitro with p87-99. Administration of p87-99[91K > A], but not p87-99 [95T > A] or p87-99[96P > A], reduced the production of tumor necrosis factor (TNF)- alpha and interferon (IFN) gamma. IFN-gamma and TNF-alpha are two cytokines that are critical in the pathogenesis of EAE and MS.

Multiple discrete encephalitogenic epitopes of the autoantigen myelin basic protein include a determinant for I-E class II-restricted T cells.

Immunization with the autoantigen myelin basic protein (MBP) causes experimental allergic encephalomyelitis (EAE). Initial investigations indicated that encephalitogenic murine determinants of MBP were located only within MBP 1-37 and MBP 89-169. Encephalitogenic T cell epitopes within these fragments have been identified. Each epitope is recognized by T cells in association with separate allelic I-A molecules. A hybrid I-E-restricted T cell clone that recognizes intact mouse (self) MBP has been examined. The epitope recognized by this clone includes MBP residues 35-47. When tested in vivo, p35-47 causes EAE. T cell recognition of p35-47 occurs only in association with I-E molecules. These results provide the first clear example that antigen-specific T cells restricted by I-E class II molecules participate in murine autoimmune disease. Furthermore, it is clear that there are multiple (at least three) discrete encephalitogenic T cell epitopes of this autoantigen, each recognized in association with separate allelic class II molecules. These results may be relevant to human autoimmune diseases whose susceptibility is associated with more than one HLA-D molecule.

Encephalitogenic T cell clones specific for myelin basic protein. An unusual bias in antigen recognition.

Class II-restricted T cell clones specific for myelin basic protein (MBP) have been generated from PL/J and (PL/J X SJL/J)F1 [((PLSJ)F1] mice following sensitization to rat MBP. Of 17 T cell clones generated from (PLSJ)F1 mice, 5 are I-Au(A alpha uA beta u) restricted, one is restricted to I-As(A alpha sA beta s), 10 are restricted to hybrid I-A(u X s)F1 (A alpha sA beta u) determinants, and one clone is restricted to hybrid I-E(u X s) (E alpha uE beta s) molecules. Thus, of 16 I-A-restricted T cell clones generated from (PLSJ)F1 mice, only one is I-As-restricted, reflecting a lack of priming to MBP in association with I-As. T cell clones restricted to I-Au and to I-E (E alpha u E beta s) molecules recognize mouse (self) MBP. Furthermore, only the five T cell clones restricted to I-Au molecules recognize a determinant in common with mouse (self) MBP within the encephalitogenic N-terminal peptide. Three such I-Au restricted T cell clones, derived independently, cause paralysis in 100% of (PL/J X SJL/J)F1 mice tested. Acute, chronic unremitting, and chronic relapsing paralysis are all induced following injection of these clones. Administration of greater numbers of cloned T cells causes acute and fatal experimental allergic encephalomyelitis, while administration of lower numbers of cloned T cells is associated with chronic unremitting and relapsing paralysis. Paralysis induced with T cell clones shares many clinical, immunologic, and histologic aspects with human demyelinating diseases such as multiple sclerosis. Histopathology reveals perivascular lymphocytic infiltration, demyelination, and remyelination. These studies demonstrate the utility of T cell clones for analyzing the association between class II major histocompatibility complex molecules and disease susceptibility.

Predominant expression of a T cell receptor V beta gene subfamily in autoimmune encephalomyelitis.

TCR beta chain gene expression of individual T cell clones that share the same MHC class II restriction and similar fine specificity for the encephalitogenic NH2 terminus of the autoantigen myelin basic protein (MBP) has been examined. TCR V beta expression was examined by FACS analysis with mAbs specific for the V beta 8 subfamily of TCR beta chain genes. 14 of 18 (78%) NH2-terminal MBP-specific clones examined express a member of the TCR V beta 8 subfamily. Southern analysis was used to identify which member(s) of the TCR V beta 8 subfamily is expressed by these clones. Each of four clones examined uses V beta 8.2, though two different V beta 8.2-J beta 2 combinations were identified. Our findings indicate that there is restricted TCR V beta usage in the autoimmune T cell response to the dominant encephalitogenic NH2-terminal epitope of the MBP. The use of an mAb to the antigen-specific TCR in the prevention of T cell-mediated autoimmune disease has been investigated. Our results demonstrate that in vivo administration of a TCR V beta 8-specific mAb prevents induction of autoimmune encephalomyelitis.

Virus-like particle capsid proteins encoded by different L double-stranded RNAs of Saccharomyces cerevisiae: their roles in maintenance of M double-stranded killer plasmids.

The plasmid determinants of killer phenotypes in type K1 and K2 killer yeast cells are the 1.9-kilobase (kb) M1 and 1.7-kb M2 double-stranded RNAs (dsRNAs), respectively. These are dependent for their maintenance and encapsidation, in Saccharomyces cerevisiae virus ScV-M1 or ScV-M2 virus-like particles, on the capsid provided by one of a group of moderately related 4.7-kb dsRNAs called LA. The L1A and L2A dsRNAs found in naturally isolated K1 and K2 killers encode 88-kilodalton VL1A-P1 and 86-kilodalton VL2A-P1 capsids, respectively. These are competent for encapsidating homologous LA dsRNAs as well as M dsRNAs. Most strains of S. cerevisiae, including killers, contain one of a second group of closely related 4.7-kb dsRNAs called LBC. These encode their own 82-kilodalton capsid protein, VLBC-P1, which, at least in strains containing only LBC, encapsidates homologous dsRNA in ScV-LBC virus-like particles. In a K1 killer strain containing both L1A and LBC, ScV-M1 particles contain only VL1A-P1. In such strains it is probable that each virus-like particle contains a single capsid type and that each L dsRNA is encapsidated by a homologous capsid.

Survival of mouse mammary gland transplants of normal, hyperplastic, and tumor tissues exposed to X-rays.

Mouse mammary tissues, including ducts, prelactating lobules, hyperplastic outgrowth lines, and tumors, were exposed to varying doses of X-rays and then transplanted to fat pads of nonirradiated BALB/c mice for study. Estimates of the dose of radiation that would allow survival of 50% of the transplants (SD50) were made with the use of probit analysis. Nearly all duct and lobule transplants survived doses of X-rays from 0 to 800 rad. The survival rate declined rapidly following doses above 800 rad, and the calculated SD50 was 1,020 and 1,260 rad for mammary ducts and lobules, respectively. The three hyperplastic outgrowth lines tested gave very different results. Hyperplastic line Z5C1 transplants had better than 90% survival at doses up to 1,200 rad and an SD50 between 1,200 and 1,600 rad. Hyperplastic line Z5D transplants had an SD50 of between 800 and 1,200 rad. Hyperplastic line D1 transplants had a better than 90% survival following doses of 0-600 rad and an SD50 between 600 and 800 rad. The survival of tumor transplants was 100% following doses of X-rays up to 1,200 rad; the SD50 was in excess of 1,600 rad. The mouse mammary transplantation system can be used to study the direct effect of X-rays on normal, premalignant, and malignant mammary tissues and provides a basis for the study of the radiobiology of mammary tissues.

Effect of dietary fat on tumorigenesis in the mouse mammary gland.

The effects of dietary polyunsaturated fat in BALB/c mice were determined for several stages of mammary tumorigenesis: a) growth of intact and transplanted normal mammary tissue, b) growth of hyperplastic alveolar nodule (HAN) lines D1/ UCD and Z5C1 , c) incidence of tumors occurring in D1/ UCD , and d) growth rate of the tumors. Growth of transplanted normal mammary epithelium as well as intact glands was faster in mice fed a 10% corn oil (CO) diet, which contains linoleate, than in those fed 10% hydrogenated cottonseed oil ( HCTO ), a diet free of the polyunsaturated fatty acid. We concluded that transplantation itself does not alter the response of mammary tissue to dietary fat. The growth of both HAN lines was unchanged by the presence of CO in the diet. For ascertainment as to whether the difference in response was specific to the tissue, ducts and HAN line D1/ UCD were transplanted into left and right gland-free fat pads of the same mice. The duct tissue grew faster when the hosts were fed the 10% CO diet than in the hosts fed the 10% HCTO diet; however, no effect of dietary fat was noted with the HAN. Although indomethacin retarded the growth of normal tissue in the mice fed CO diet, this prostaglandin inhibitor had no effect on the growth of the HAN transplants. Both incidence and growth rate of tumors arising from transplants of HAN line D1/ UCD were greater in mice fed diets containing either 0.3, 1, or 10% CO than in those fed 10% HCTO . The smaller number of tumors arising in mice fed with 10% HCTO diet may be the result of the slower growth rate of the neoplasms in mice fed diets devoid of linoleate.

Hyperplastic and neoplastic changes in the mammary glands of feral mice free of endogenous mouse mammary tumor virus provirus.

Laboratory colonies of feral mice (Mus musculus domesticus) have been established with specific mouse mammary tumor virus (MuMTV) genotype, including colonies lacking any proviral DNA (ev-) or carrying only a single copy of MuMTV DNA (ev+). No evidence of a decline in reproductive capacity has been observed in the first 8 generations. Both the ev- and ev+ mice showed normal mammary gland development and the development of hyperplastic lesions in the older females. The mice were very resistant to spontaneous or chemically induced mammary tumors. However, the occurrence of 1 mammary tumor in an ev- mouse indicates that mammary neoplasias can occur in the absence of MuMTV DNA. The few tumors that do occur in the ev- mice provide a unique opportunity to study the neoplastic process in the absence of proviral DNA.

Development and characterization of mouse hyperplastic mammary outgrowth lines from BALB/cfC3H hyperplastic alveolar nodules.

Five hyperplastic outgrowth lines were developed by serial transplantation of hyperplastic alveolar nodules from BALB/cfC3H mice into BALB/c hosts. The lines were used to study the biology, morphology, and virology of premalignant tissue originating in mouse mammary tumor virus (MuMTV)-positive animals. The five lines differed with respect to tumor potential and growth characteristics, corroborating the previous evidence that MuMTV-positive hyperplastic alveolar nodules are biologically heterogeneous. Subgross and microscopic examination of outgrowths and tumors revealed that each line had unique morphological characteristics. The presence of atypical lobules within the hyperplastic outgrowth appeared to be correlated with tumor risk, and a morphological continuum of atypical lesions ending in overt cancer was suggested. Viral expression was detected by nucleic acid hybridization and immunoperoxidase staining for MuMTV structural antigens. While the MuMTV RNA in certain tissues appeared to vary qualitatively with tumor potential of the outgrowth line, no correlation between viral antigens detected by immunoperoxidase staining and tumor potential was observed.

A model for the packing of lipids in bilayer membranes.

A number of known structural properties of mixed lipid bilayer membranes and monolayers are accounted for by a model in which lipids pack into bilayers and monolayers like building blocks, each characterized by a surface head group area and characteristic solid angle. In phospholipids above the melting transition the head group area (at a given temperature and degree of hydration) is fairly invariant while the hydrocarbon region may be liquid-like so long as the molecule is not compressed beyond its characteristic solid angle. Phosphatidylcholine and phosphatidylserine are tapered lipids, i.e. their surface head group areas are greater than their non-polar end areas; cholesterol is frayed, i.e. its polar end area is less than its non-polar end area; while phosphatidylethanolamine is almost cylindrical. The "condensing" effect of cholesterol in mixed phospholipid-cholesterol films is seen as a taper-fray accommodation. The lipid distribution in erythrocyte membrane is shown to be conducive to a stable strain-free membrane.

Phase transition in charged lipid membranes.

Experimental results on the effect of electrostatics on bilayer phase transitions are compared with corresponding data for monolayers and the predictions of electrical double layer theory. The two substantial conclusions which emerge are that: (i) double layer theory based on a continuous surface charge distribution cannot explain all the relevant data, a situation which may be improved by taking into account the discrete nature of the surface charge distribution; (ii) the crystal - liquid crystal phase transition of charged bilayer membranes is always a continuous one which takes place through an intermediate state consisting of both fluid and frozen domains.

Theory of self-assembly of lipid bilayers and vesicles.

A simple theory is developed that explains the formation of bilayers and vesicles and accounts quantitatively for many of their physical properties: Properties including vesicle size distributions and bilayer elasticity emerge from a unified theory that links thermodynamics, interaction free energy, and molecular geometry. The theory may be applied to the analysis of more complicated membrane structures and mechanisms.

First Report of Pythium Root Rot of Rau Ram (Polygonum odoratum).

Polygonum odoratum (= Persicaria odorata), known as rau ram or sang hum, is native to southeastern Asia and is a common herb in Vietnamese cuisine (1). It has been studied most extensively for its aromatic compound content (2). In Florida, rau ram commonly is grown hydroponically in greenhouses using large, cement beds with recirculated water. The plants form dense mats from which new growth is trimmed for market. During January of 2002, a severe dieback was observed in one production house in Saint Lucie County, FL. Plants with less severe symptoms were yellowed and stunted. Roots of symptomatic plants were largely decayed with root symptoms beginning as a tip necrosis. The cortex of severely affected roots slipped off easily, leaving a stringy vascular system. Plating of symptomatic tissue from 20 randomly selected plant samples was performed with multiple general and selective media including potato dextrose agar, corn meal agar with pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP) (3). All colonies produced were identified as Pythium helicoides Drechsler on the basis of sporangial, oogonial, and antheridial characteristics (4). Isolates had proliferous, obovoid, papillate sporangia, and were homothallic with smooth-walled oogonia and thick-walled, aplerotic oospores. Multiple antheridial attachments per oogonium were common with the antheridium attached along its entire length. Pathogenicity tests were conducted using P. odoratum plants grown from commercial transplants. Two tests were performed. Each test was conducted using eight inoculated and eight control plants. In the first test, plants were maintained in 10-cm pots immersed in sterilized pond water for the duration of the test. Plants were inoculated with five 7- × 70-mm sections of freshly growing mycelial culture per plant using 10-day-old cultures of Pythium helicoides grown on water agar. Chlorosis was observed at approximately 2 months after inoculation. Root necrosis was observed in inoculated plants approximately 5 months after inoculation. This test was performed in the greenhouse with temperatures ranging from 20 to 30°C. The second test was performed in growth chambers at 35 to 40°C. Plants were maintained in 10-cm pots immersed in Hoagland's solution and were inoculated with four 6-mm plugs per plant. Symptoms were observed on inoculated plants at this temperature within 1 week of inoculation. No chlorosis or root decay was observed in noninoculated, immersed plants. The pathogen was reisolated from inoculated, symptomatic tissue. To our knowledge, this is the first report of root rot of P. odoratum caused by Pythium helicoides. References: (1) R. E. Bond. Herbarist 55:34, 1989. (2) N. X. Dung et al. J. Essent. Oil Res. 7:339, 1995. (3) M. E. Kannwischer and D. J. Mitchell. Phytopathology 68:1760, 1978. (4) A. J. van der Plaats-Niterink. Monograph of the Genus Pythium. Vol. 21, Studies in Mycology. Centraalbureau voor Schimmelcutltures, Baarn, The Netherlands, 1981.

Unusual G + C content and codon usage in catIJF, a segment of the ben-cat supra-operonic cluster in the Acinetobacter calcoaceticus chromosome.

The nucleotide (nt) sequence of a 5.3-kb DNA segment containing the Gram- Acinetobacter calcoaceticus catBCIJFD operon is reported. This information completes determination of a 16-kb nt sequence containing the twelve ben and cat structural genes encoding enzymes required for catabolism of benzoate via the beta-ketoadipate pathway. Many of these genes can be traced to a common ancestry with genes from other organisms containing DNA with widely divergent G + C content. The A. calcoaceticus ben and cat genes are arranged in a supra-operonic cluster containing one known regulatory gene and three additional open reading frames (ORFs) that may have regulatory functions. Thirteen of the ben and cat genes, including the three ORFs with unknown function, are typical for A. calcoaceticus in that they possess a G + C content of 44.9 +/- 2.5%. Three exceptional A. calcoaceticus genes (catI, catJ and catF) possess G + C contents of 56.5 +/- 1.3%. These differences in G + C content are reflected in the distinctive patterns of codon usage shared by catI, catJ and catF. Thus, the catIJF region, known to exchange genetic information with the pcaIJF region in the same chromosome directing isofunctional proteins associated with the beta-ketoadipate pathway, has avoided the evolutionary forces that conferred characteristics G + C content upon the other ben and cat genes in A. calcoaceticus.

Phase 1 clinical trial of chimeric monoclonal anti-CD4 antibody in multiple sclerosis.

We conducted an open trial of cM-T412, a chimeric monoclonal anti-CD4 antibody, in 29 patients with MS. This antibody caused a prompt and long-lasting depletion of circulating CD4 (helper/inducer) lymphocytes. The mean (+/- SE) CD4 count for the group decreased from 870 (+/- 66) cells/mm3 at baseline to 76 (+/- 11) 3 hours after treatment, and then increased to 425 (+/- 38) at 1 month after treatment and 475 (+/- 39) at 6 months after treatment. Numbers of CD8 (cytotoxic/suppressor) lymphocytes, B lymphocytes, granulocytes, and monocytes changed transiently but showed no significant long-term effects. The most common side effects were headache, nausea, myalgia, fever, and tachycardia occurring in the first few hours after treatment. No serious or unexpected infections or other significant adverse effects occurred. Kurtzke EDSS scores remained stable, and MRI scans showed less contrast enhancement 1 week after treatment. We conclude that treatment of MS patients with cM-T412 chimeric anti-CD4 antibody is well tolerated at the doses tested and produces a long-lasting, selective depletion of CD4 lymphocytes.

Characterization of a major encephalitogenic T cell epitope in SJL/J mice with synthetic oligopeptides of myelin basic protein.

The C-terminal 89-169 amino acid fragment of myelin basic protein (MBP) causes experimental allergic encephalomyelitis (EAE) in SJL/J mice. In order to identify the encephalitogenic T cell epitope, we have examined the fine specificity of encephalitogenic SJL/J T cell clones with synthetic peptides derived from the C-terminal 89-169 amino acids of MBP. These peptides were examined for their immunogenic and encephalitogenic activity in the SJL/J mouse. The SJL/J-derived, encephalitogenic T cell clone, 4b.14a, was shown to be responsive to rat myelin basic protein synthetic peptides pR89-101 (VHFFKNIVTPRTP) as well as to intact MBP. Its response was effectively blocked by mAb 10-2.16 (anti-I-As) as was the response to intact MBP. Furthermore, pR89-101 was revealed to be highly immunogenic for the (PLSJ)F1 mouse in terms of lymphocyte proliferation, but not for the PL/J mouse, in spite of the fact that there exists a strong bias to H-2u restricted responses in the (PLSJ)F1 mouse at the T cell level. By using pR89-101, T cells of (PLSJ)F1 origin were revealed to recognize the peptide in association with the I-As molecule on (PLSJ)F1 antigen presenting cells (APC). When examined for encephalitogenicity for the SJL/J mouse, pR89-101 was found to be as encephalitogenic as intact rat MBP. These results demonstrated that MBP peptide pR89-101 is a major encephalitogenic determinant for the SJL/J mouse.

Is integer arithmetic fundamental to mental processing?: the mind's secret arithmetic.

Unlike the ability to acquire our native language, we struggle to learn multiplication and division. It may then come as a surprise that the mental machinery for performing lightning-fast integer arithmetic calculations could be within us all even though it cannot be readily accessed, nor do we have any idea of its primary function. We are led to this provocative hypothesis by analysing the extraordinary skills of autistic savants. In our view such individuals have privileged access to lower levels of information not normally available through introspection.

T cell recognition in experimental autoimmune encephalomyelitis: prospects for immune intervention with synthetic peptides.

Peptide binding and lymph node T cell activation studies have been used to characterize T cell recognition of an encephalitogenic T cell autoantigen from myelin basic protein in mice of the H-2u haplotype. An important role for MHC class II molecules in "determinant selection" is revealed. Amino acids which determine interactions with either the restriction element of the major histocompatibility complex (MHC) or the encephalitogenic T cell receptor are defined. This information enables the design of peptides which bind MHC yet do not crossreact with the autoantigen. Two such peptides compete with the autoantigen for binding to the disease associated class II molecule and inhibit induction of experimental autoimmune encephalomyelitis in H-2u mice. Prospects for peptide mediated therapy are discussed.

Effects of X irradiation on the growth of normal and hyperplastic mouse mammary gland transplants.

To avoid the problems associated with whole-body radiation, pieces of X-irradiated normal or hyperplastic mammary tissue were transplanted to the host gland-free fat pad of nonexposed mice. The percentage of the fat pad filled by growth of the transplants at 4, 8, and 12 weeks after transplantation was measured. Growth of lobule transplants was moderately inhibited by 4 Gy. While some of the lobules survived 12 Gy, their growth was severely inhibited. The hyperplastic outgrowth lines were variable but more resistant than lobules to growth retardation. Line Z5D was more susceptible than D1, and Z5C1 was least susceptible, with 88% growing well after 12 Gy. In order to distinguish between transient and permanent growth retardation, tissue was taken from the irradiated and control transplants and retransplanted to new hosts without further radiation. The second generation of X-ray-exposed tissue filled more of the fat pad than the first-generation transplants, but significantly less than the nonexposed controls. The experiments described provide a means of demonstrating X-ray-induced changes in the mammary gland from growth inhibition to carcinogenesis.

Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA.

The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Pythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual cultures or from mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi.