Induction of experimental allergic neuritis in the BN rat: P2 protein-specific T cells overcome resistance to actively induced disease

T lymphocyte lines specific for the peripheral nerve myelin protein P2 were selected from the lymph nodes of Brown Norway (BN) rats immunized with bovine P2 protein in complete Freund's adjuvant. These T cells expressed the W3/25+, OX8-phenotype and responded specifically to bovine P2 protein, but not to PPD or bovine basic protein, in T cell proliferation assays. When injected i.v. into syngeneic recipients, BN P2-specific T cell lines induced both clinical and histologic signs of experimental allergic neuritis (EAN), overcoming the resistance of this rat strain to actively induced EAN. Although the histopathology of the disease was indistinguishable from that seen in T cell-mediated EAN in the Lewis rat, disease onset was considerably later, 7 to 8 days after cell transfer, as opposed to 4 days in Lewis. This lag phase between inoculation and disease onset could not be further reduced even by raising the cell dose to 50 X 10(6) cells/host. The fine specificity of the T cell response to P2 differs between Lewis- and BN-derived T cell lines. At least one neuritogenic epitope for each strain was present in the cyanogen bromide-derived peptide CB2 (residues 21-113), as shown by the ability of CB2-specific T cell lines derived from each strain to transfer EAN to the appropriate host strain. However, neuritogenic BN T lines fail to mount a response to the sequence 53-78 (SP4), which encompasses an epitope that is neuritogenic for Lewis rats. These results demonstrate that the resistance of BN rats to actively induced EAN is not due to the lack of appropriate P2-specific autoreactive T cell clones in the normal T repertoire. Furthermore, the results suggest that two distinct epitopes of P2 are responsible for EAN in Lewis and BN rats.     

A permanent rat T cell line that mediates experimental allergic neuritis in the Lewis rat in vivo

A rat T cell line of the "helper" phenotype (W3/25-positive, OX 8-negative) has been derived from Lewis rats inoculated with P2 protein isolated from bovine PNS myelin. The line LiP2/A is exquisitely specific for P2 protein, exhibiting no reactivity to bovine basic protein or to PPD. In addition to responding strongly to the intact P2 protein, the line cells show some response to a synthetic peptide containing the neuritogenic amino acid sequence of P2 protein (SP-B, residues 66-78). Intravenous inoculation of naive rats with as few as 10(4) activated LiP2/A cells leads to the onset of mild clinical signs of experimental allergic neuritis. Higher doses of cells lead to more severe clinical disease. Histologic examination of clinically ill animals confirmed the disease as EAN. The pathologic lesions were confined to the PNS and spared the central nervous system. The lesions consisted of marked perivascular cuffs and infiltrates of inflammatory cells associated with marked degenerative changes--demyelination and some axonal degeneration.     

The passive transfer of severe allergic neuritis in Lewis rats with lymphoid cells preincubated with P2 protein

The passive transfer of both clinical signs and histologic lesions characteristic of allergic neuritis was successfully performed in Lewis rats using pooled spleen and lymph node cells, or T lymphocytes therefrom, if first preincubated in petri dishes with P2 protein for 72 hr. For passive transfer, cells were taken from donors 8-16 days after sensitization with P2 protein or myelin in Freund's complete adjuvant, and administered via the tail vein; clinical signs appeared 12-13 days later. This study supports the importance of cell-mediated immunity in EAN and the antigenic role of the P2 protein.     

Transfer of experimental allergic neuritis with P2-reactive T-cell lines

Experimental allergic neuritis (EAN) was induced in normal Lewis rats by systemic passive transfer of T-cell lines responding to P2 protein. These cells had predominantly helper phenotype and could induce EAN within 7 days following adoptive transfer. There was no anti-P2 antibody response in the recipients of the P2-reactive cells recovered from donors with high anti-P2 antibody levels. This study provides direct evidence that T cells are important for the induction of EAN. Furthermore, there was no evidence of a pathogenic role for anti-P2 antibody in passive EAN.     

Prevention and therapy of experimental autoimmune neuritis by an antibody against T cell receptors-alpha/beta.

The mAb R73 directed to the TCR-alpha/beta of rat lymphocytes was tested for its therapeutic potential during the effector phase of experimental autoimmune neuritis (EAN) in Lewis rats. EAN can be actively induced by immunization with bovine peripheral nerve myelin, bovine P2 protein, or a peptide containing its neuritogenic epitope and serves as a model of the human Guilain-Barré syndrome. Adoptive transfer of activated P2-specific T lymphocytes also produces the monophasic disease (AT-EAN) characterized by inflammation and demyelination of peripheral nerves and highlights the central role of T lymphocytes in the pathogenesis of EAN. A single administration of the mAb R73 immediately after injection of activated P2-specific T line cells completely prevented the development of clinical and electrophysiologic signs of EAN in most animals and greatly alleviated the disease in the others. In further experiments mAb R73 was applied after the appearance of first clinical signs of EAN actively induced by immunization with a neuritogenic peptide or bovine peripheral nerve myelin. In both cases the anti-TCR-alpha/beta mAb reversed clinical signs of EAN and prevented the development of peripheral nerve dysfunction. In vivo and in vitro data suggest that impairment of Ag recognition and T cell function by occupancy of the TCR and R73-induced TCR-modulation rather than depletion of TCR-alpha/beta-bearing lymphocytes is the decisive mechanism underlying suppression of EAN that is apparent already within 48 h of the first R73 injection.     

Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of liposomes by type II cells

The alveolar type II cell which synthesizes and secretes surfactant also plays a major role in the reuptake of surfactant lipids. In a recent in vivo study we found that the subfractions of natural surfactant that contained the surfactant protein with molecular weights of 26,000-36,000 (SP-26-36) were preferentially taken up into lamellar bodies of type II cells to a greater extent than were fractions that did not contain SP-26-36. Because the subfractions of natural surfactant in that study differed in other properties than the presence or absence of SP-26-36, the current study was undertaken to determine whether purified SP-26-36 enhanced the uptake of surfactant-like lipids by freshly isolated type II cells. SP-26-36 increased the uptake of label in radioactive surfactant-like lipids by up to 10-fold, and the effect of SP-26-36 was dependent on time, protein concentration, and temperature. The enhancement was inhibited by heat-treating the protein, by a polyclonal antibody against SP-26-36, and by metabolic inhibitors. The distribution of radioactivity in cell-associated phospholipids differed if cells were incubated with or without SP-26-36. If SP-26-36 was present during the incubation, greater than 96% of the radioactivity remained associated with phosphatidylcholine. In the absence of SP-26-36, only 85% of the radioactivity remained associated with phosphatidylcholine and 7% of the label appeared in phosphatidylglycerol. We hypothesize that SP-26-36 may act as a ligand to direct surfactant lipids to type II cells, perhaps to different metabolic pathways, and to regulate recycling and surfactant homeostasis.     

Neuritogenic Lewis rat T cells use Tcrb chains that include a new Tcrb-V8 family member

The P2 protein obtained from Schwann cells induces a population of T cells which, upon adoptive transfer, causes the disease experimental allergic neuritis (EAN), an animal model for Guillain-Barre syndrome. In this report, a truncated peptide, FR22, derived from a previously reported neuritogenic T-cell determinant, was used to generate from Lewis rats T cells that were shown to cause EAN. Since our previous studies showed that Tcrb-V8 was used by a majority of T-cell hybridomas specific for the neuritogenic peptide P26, which contains the FR22 sequence, we sequenced the Tcrb-V8 ⁺ mRNA from FR22-specific T-cell lines, and compared the sequences obtained with those obtained from similarly generated myelin basic protein (MBP) 68–88-specific Lewis rat T-cell lines. We found that in the EAN lines, several members of the Tcrb-V8 family were used, including a new family member, Tcrb-V8E. This was more diverse than the MBP-68–88-specific response in which only a single Tcrb-V8 family member was used. Also, in the EAN lines, the beta chain sequences did not show the same conserved junctional regions seen in the MBP lines. Thus, T-cell receptor beta chain usage in the response to this dominant neuritogenic peptide appears to be less restricted than the response to the dominant encephalitogenic determinant of MBP both in V region usage and in CDR3 usage.The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databse and have been assigned the accession numbers UO6100 (Tcrb-V8A), UO6101 (Tcrb-V8B), UO6102 (Tcrb-V8C), UO6103 (Tcrb-V8D), and UO6104 (Tcrb-V8E)     

A complementary peptide vaccine that induces T cell anergy and prevents experimental allergic neuritis in Lewis rats.

We have developed and described a new method of altering T cell-mediated autoimmune diseases by immunization with the complementary peptide against T cell epitopes. The complementary peptide (denoted NAE 07-06) to the bovine P2 protein, residues 60-70 (denoted EAN 60-70), was tested in the Lewis rat model of experimental allergic neuritis (EAN). Immunization with NAE 07-06 induced polyclonal and monoclonal Abs that inhibited the proliferation of the P2-specific T cell line, stimulated with EAN 60-70, and recognized Vbeta, but not Valpha, of TCRs. Proliferation of T cells treated with anti-NAE 07-06 Abs could be partially restored by treatment with rIL-2, in accordance with an anergy model. A homologous sequence was found between NAE 07-06 and the VDJ junction of the TCR beta-chain from an EAN 60-70-specific T cell line. Rats preimmunized with NAE 07-06 in vivo before EAN induction showed less disease severity clinically and histologically. These data suggest a new therapeutic approach for T cell-mediated autoimmune disorders through the induction of anti-TCR Abs with complementary peptide Ags.     

Attenuated EAN in TNF-α deficient mice is associated with an altered balance of M1/M2 macrophages

The role of tumor necrosis factor (TNF)-α and its receptors in neuroautoimmune and neuroinflammatory diseases has been controversial. On the basis of our previous studies, we hereby aimed to further clarify TNF-α's mechanism of action and to explore the potential role of TNF-α receptor (TNFR)1 as a therapeutic target in experimental autoimmune neuritis (EAN). EAN was induced by immunization with P0 peptide 180-199 in TNF-α knockout (KO) mice and anti-TNFR1 antibodies were used to treat EAN. Particularly, the effects of TNF-α deficiency and TNFR1 blockade on macrophage functions were investigated. The onset of EAN in TNF-α KO mice was markedly later than that in wild type (WT) mice. From day 14 post immunization, the clinical signs of TNF-α KO mice were significantly milder than those of their WT counterparts. Further, we showed that the clinical severity of WT mice treated with anti-TNFR1 antibodies was less severe than that of the control WT mice receiving PBS. Nevertheless, no difference with regard to the clinical signs of EAN or inflammatory infiltration in cauda equina was seen between TNF-α KO and WT mice with EAN after blockade of TNFR1. Although TNF-α deficiency did not alter the proliferation of lymphocytes in response to either antigenic or mitogenic stimuli, it down-regulated the production of interleukin (IL)-12 and nitric oxide (NO), and enhanced the production of IL-10 in macrophages. Increased ratio of regulatory T cells (Tregs) and reduced production of interferon (IFN)-γ in cauda equina infiltrating cells, and elevated levels of IgG2b antibodies against P0 peptide 180-199 in sera were found in TNF-α KO mice with EAN. In conclusion, TNF-α deficiency attenuates EAN via altering the M1/M2 balance of macrophages.     

The spirostenol (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate blocks mitochondrial uptake of Abeta in neuronal cells and prevents Abeta-induced impairment of mitochondrial function

Abeta(1-42) has been shown to uncouple the mitochondrial respiratory chain and promote the opening of the membrane permeability transition (MPT) pore, leading to cell death. We have previously reported that the spirostenol derivative (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate (SP-233) protects neuronal cells against Abeta(1-42) toxicity by binding to and inactivating the peptide. Picomolar concentrations of Abeta(1-42) decreased the mitochondrial respiratory coefficient in mitochondria isolated from the rat forebrain, and this decrease was partially reversed by SP-233. SP-233 abolished the uncoupling of oxidative phosphorylation induced by carbonyl cyanide 3-chlorophenylhydrazone on isolated mitochondria. These results are consistent with a direct effect of SP-233 on the MPT. Moreover, SP-233 displayed a neuroprotective effect on SK-N-AS human neuroblastoma cells treated with the MPT promoter, phenylarsine oxide. Treatment of SK-N-AS cells with Abeta(1-42) resulted in an accumulation of the peptide in the mitochondrial matrix; SP-233 completely scavenged Abeta(1-42) from the matrix. In addition, SP-233 protected the cells against mitochondrial toxins targeting complexes IV and V of the respiratory chain. These results indicate that Abeta(1-42) and SP-233 exert direct effects on mitochondrial function and SP-233 protects neuronal cells against Abeta-induced toxicity by targeting Abeta directly.     

Forced-exercise attenuates experimental autoimmune neuritis

Physical inactivity in combination with a sedentary lifestyle is strongly associated with an increased risk of development of inflammatory-mediated diseases, including autoimmune disorders. Recent studies suggest that anti-inflammatory effects of physical exercise may be of therapeutic value in some affected individuals. In this study, we determined the effects of forced-exercise (treadmill running) on the development and progression of experimental autoimmune neuritis (EAN), an established animal model of Guillain-Barré syndrome. Adult male Lewis rats were subjected to sedentary (control) or forced-exercise (1.2 km per day, 5 days a week) for three weeks prior to induction of EAN. P2 (53-78)-immunized sedentary control rats developed a monophasic course of EAN beginning on post-injection day 12.33 ± 0.59 (n = 18) and reaching peak severity on day 15.83 ± 0.35 (n = 18). At near peak of disease, ankle- and sciatic notch-evoked compound muscle action potential (CMAP) amplitudes in sedentary control rats were reduced (~50%) while motor nerve conduction velocity (MNCV) was slowed (~30%) compared with pre-induction evoked responses. In marked contrast, rats undergoing forced-exercise exhibited a significantly less severe clinical course of EAN beginning on post-injection day 12.63 ± 0.53 (n = 16) and reaching peaking severity on day 14.69 ± 0.73 (n = 16). At near peak of disease, ankle- and sciatic-notch-evoked CMAP amplitudes in forced-exercised rats were preserved while EAN-associated slowing of MNCV was modestly attenuated by exercise. Three weeks of forced-exercise reduced by 46% total plasma corticosterone content while elevating the levels of corticosteroid binding globulin. We conclude from this study that forced-exercise administered prior to and during development of EAN affords a novel measure of protection against autoimmune-associated deficits in peripheral nerve evoked responses independent of steroid-induced immune suppression.     

Mast cell proteases liberate stable encephalitogenic fragments from intact myelin.

Protease-containing supernatants from activated rat mast cells were found to degrade purified rat myelin with a subsequent release of a stable encephalitogenic peptide. The two most abundant peptides were identified as residues 69-87 (GSLPQKSQRTQDENPVV) and residues 69-88 (GSLPQKSQRTQDENPVVH). While additional exposure to the mast cell supernatants removes the COOH terminal histamine from peptide 69-88 to yield peptide 69-87, additional proteolytic degradation of the 69-87 peptide was not detected. Immunization with this peptide emulsified in CFA caused the development of clinical experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In addition this 69-87 sequence was found to activate resting encephalitogenic myelin basic protein-reactive T cell lines to adoptively transfer clinical EAE. The release of stable encephalitogenic peptides from the myelin sheath by mast cell proteases may play a role in activation of encephalitogen-specific T cells during the progression of EAE.     

Spontaneous development of protective anti-T cell receptor autoimmunity targeted against a natural EAE-regulatory idiotope located within the 39-59 region of the TCR-V beta 8.2 chain.

The development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats is mediated by V beta 8.2+ T cells specific for myelin basic protein. One consequence of this biased expression of V beta 8.2 is the spontaneous development of regulatory T cells and antibodies against residues 39-59 of the V beta 8.2 sequence. Moreover, a synthetic V beta 8.2-39-59 peptide could induce protection against and speed recovery from EAE. T cells and antibodies specific for V beta 8.2-39-59 could transfer protection from EAE. Recently, we reported that the protective T cell epitope is subsumed within the V beta 8-44-54 sequence. We now report that protection induced by V beta 8-44-54 lasted at least 102 days and produced "split tolerance," enhancing anti-myelin basic protein antibody titers but reducing anti-myelin basic protein T cell frequency. The shorter V beta 8-44-54 peptide induced a distinct set of antibodies that did not cross-react with the longer V beta 8.2-39-59 peptide, although both specificities could stain V beta 8.2+ T cells and were equally protective against EAE. However, the V beta 8.2-39-59 peptide, but not the V beta 8-44-54 peptide, would appear to represent the natural idiotope: antibodies to V beta 8.2-39-59 that develop spontaneously during EAE could be boosted to higher titers only by the V beta 8.2-39-59, but not by other TCR peptides from the V beta 8.2 sequence, including V beta 8-44-54 that contains the functional T cell epitope. These results suggest that natural processing of the TCR V beta-chain favors the formation of a peptide that resembles the V beta 8.2-39-59 sequence. The B cell epitope present on the V beta 8-44-54 sequence was evident only in the absence of residues 39-43 and 55-59, suggesting that the two peptides possess distinct conformations. However, the V beta 8-44-54 B cell epitope is most likely expressed on the V beta 8.2+ T cells, either as a low affinity determinant on the intact TCR alpha/beta heterodimer or as a cryptic epitope bound in the cleft of surface MHC molecules.     

Characterization of the antigen specificity and TCR repertoire,and TCR-based DNA vaccine therapy in myelin basic protein-induced autoimmune encephalomyelitis in DA rats

Like Lewis rats, DA rats are an experimental autoimmune encephalomyelitis (EAE)-susceptible strain and develop severe EAE upon immunization with myelin basic protein (MBP). However, there are several differences between the two strains. In the present study we induced acute EAE in DA rats by immunization with MBP and MBP peptides and examined the Ag specificity and TCR repertoire of encephalitogenic T cells. It was found that although immunization with MBP and a peptide corresponding to its 62-75 sequence (MBP(62-75)) induced clinical EAE, the responses of lymph node T cells isolated from MBP-immunized rats to MBP(62-75) was marginal, indicating that this peptide contains major encephalitogenic, but not immunodominant, epitopes. The TCR analysis by CDR3 spectratyping of spinal cord T cells revealed that Vbeta10 and Vbeta15 spectratype expansion was always found in MBP(62-75)-immunized symptomatic rats. On the basis of these findings, we examined the encephalitogenicity of Vbeta10- and Vbeta15-positive T cells. First, the adoptive transfer experiments revealed that Vbeta10-positive T line cells derived from MBP(62-75)-immunized rats induced clinical EAE in recipients. Second, administration of DNA vaccines encoding Vbeta10 and Vbeta15, alone or in combination, ameliorated MBP(62-75)-induced EAE. Collectively, it was strongly suggested that Vbeta10- and Vbeta15-positive T cells are encephalitogenic. Analyses of the Ag specificity and T cell repertoire of pathogenic T cells performed in this study provide useful information for designing specific immunotherapies against autoimmune diseases.     

Selection and characterization of colorectal cancer cell-specific peptides

To develop a novel peptide probe for imaging detection of colorectal cancer, a 12-mer phage display library was used to select peptides that bind specifically to the human colorectal cancer cell line Caco-2. After four rounds of panning, four phage clones that bound specifically to the Caco-2 cells were selected. The phage clone SP-2 had a particularly high affinity and specificity for Caco-2 cells. This clone was identified using a series of methods. The peptide SP-2 that was displayed on phage SP-2 exhibited a high specificity to Caco-2 cells. Thus, the peptide SP-2 could be a candidate probe for the detection of colorectal cancer.     

Fish cell culture: Characteristics of a cell line from the silver perch,Bairdiella chrysura

Summary A cell line designated SP-1 was established from tissue of the silver perch,Bairdiella chrysura. Cells were fibroblast-like and grew best at 26°C in Leibovitz medium (L-15) containing 15% fetal bovine serum and 0.150m sodium chloride. Passage 1 to passage 9 SP-1 cells contained a chromosome number of 48; at passages 27 and 50 the modal numbers were 51 and 54, respectively. Confirmation of the origin of SP-1 cells was made by the cytotoxic antibody dye-exclusion test. This cell line supported the growth of lymphocystis virus from the silver perch but was not found to replicate various other fish and mammalian viruses. This study was supported in part by fund supplied by the Faculty Research Council of the University of Southern Mississippi and by the National Oceanographic and Atmospheric Administration, Office of Sea Grant No. 04-3-158-58. A portion of these results were presented at the 26th Annual Meeting of the Tissue Culture Association, Motreal, Canada, 1975.     

The immunopathology of acute experimental allergic encephalomyelitis induced with myelin proteolipid protein. T cell receptors in inflammatory lesions.

To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.     

Genetic control of the development of experimental allergic encephalomyelitis in rats. Separation of MHC and non-MHC gene effects

Experimental allergic encephalomyelitis (EAE)-susceptible Lew and EAE-resistant Brown Norway (BN) rats and the corresponding MHC congenic strains were examined for their ability to develop clinical and histologic EAE. The ability of T cells from these animals to proliferate in vitro in response to whole guinea pig (GP) myelin basic protein (MBP), rat MBP, and to the major encephalitogenic peptide of GP MBP 66-88 (GP 68-88) was also assessed. We found that Lewis (Lew) was highly susceptible and showed good T cell responses to GP, MBP, rat MBP, and GP 68-88. Lew.1N (BN MHC on Lew background) and BN were not susceptible and T cells from these strains showed significant responses to GP MBP, but not to rat MBP or GP 68-88. Although BN.B1 (Lew MHC on BN background) was not susceptible to actively induced EAE, MBP-specific Lew T cells could transfer severe disease to BN.B1. BN.B1 T cells showed responses to GP-MBP, rat MBP, and GP 68-88 and, when transferred to naive BN.B1 or Lew, induced only mild clinical EAE in both strains. Increasing the number of T cells from BN.B1 had no effect on the severity of clinical symptoms in either recipient, suggesting some deficiency in the T cell repertoire that is necessary for induction of severe EAE. These results suggest that 1) the T cell response to rat MBP and GP68-88 (but not to sites other than 68-88 in GP MBP) is necessary for susceptibility to EAE; 2) the ability to respond to both rat MBP and GP 68-88 is determined by the MHC gene products on APC; and 3) given a permissive MHC, the T cell response that results in EAE is influenced by non-MHC genes.     

A 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin perturbs the blood-testis barrier and disrupts spermatogenesis reversibly in vivo.

When Sertoli cells were cultured in vitro on Matrigel-coated bicameral units, the assembly of the inter-Sertoli tight junction (TJ) permeability barrier correlated with an induction of occludin expression. Inclusion of a 22-amino acid peptide, NH(2)-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to residues 209-230 in the second extracellular loop of rat occludin, at 0.2-4 microM into Sertoli cell cultures could perturb the assembly of Sertoli TJs dose-dependently and reversibly. This peptide apparently exerts its effects by interfering with the homotypic interactions of two occludin molecules between adjacent Sertoli cells at the sites of TJs, thereby disrupting TJs, which, in turn, causes a decline in transepithelial electrical resistance across the Sertoli cell epithelium. When similar experiments were performed using a 22-amino acid myotubularin peptide, NH(2)-TKVNERYELCDTYPALLAVPAN-COOH (residues 156-177), no effects on the assembly of inter-Sertoli TJs in vitro were noted. When a single dose of this synthetic occludin peptide was administered to adult rats intratesticularly at 1.5-10 mg/testis, germ cells began to deplete from the seminiferous epithelium within 8-16 days. By 27 days, virtually all tubules were devoid of germ cells. This antispermatogenic effect was reversible, because germ cells progressively repopulated the epithelium thereafter. Treated testes were indistinguishable from normal or control testes by 68 days post-occludin peptide treatment when assessed using histological analysis. In contrast, control rats receiving either no treatment, vehicle alone, or a 22-amino acid synthetic peptide of myotubularin displayed no changes in the testicular morphology at all time points. The occludin peptide-induced germ cell depletion was also accompanied by a disruption of the blood-testis barrier (BTB) when assessed by micropuncture techniques quantifying [(125)I]-BSA in rete testis fluid and seminiferous tubular fluid following i.v. administration of [(125)I]-BSA through the jugular vein. These results illustrate that the occludin peptide-induced disruption of the BTB may possibly affect the underlying adherens junctions, which causes premature release of germ cells from the epithelium and reversible infertility.     

Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice

PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.