Paul D. Crowe

Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo- controlled, randomized phase II trial

In this ‘double-blind’, randomized, placebo-controlled phase II trial, we compared an altered peptide ligand of myelin basic protein with placebo, evaluating their safety and influence on magnetic resonance imaging in relapsing–remitting multiple sclerosis. A safety board suspended the trial because of hypersensitivity reactions in 9% of the patients. There were no increases in either clinical relapses or in new enhancing lesions in any patient, even those with hypersensitivity reactions. Secondary analysis of those patients completing the study showed that the volume and number of enhancing lesions were reduced at a dose of 5 mg. There was also a regulatory type 2 T helper-cell response to altered peptide ligand that cross-reacted with the native peptide.

A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin.

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.

CRF receptor type 1 and 2 expression and anatomical distribution in the rat colon.

Corticotropin‐releasing factor (CRF) receptor agonists administered peripherally increase colonic propulsive motility and fecal output in experimental animals. In addition, endogenous CRF‐related peptides are found in the lower gastrointestinal (GI) tissues, suggesting a local expression of CRF receptors. In the present study, we report the expression of both CRF receptor type 1 (CRF1) and 2 (CRF2) in the rat colon at the mRNA and protein levels. For the purpose of receptor protein mapping, a specific antiserum against the C‐terminus of CRF2 (2064a‐CRF2) was generated and characterized. This antiserum in conjunction with a selective anti‐CRF1 antiserum (4467a‐CRF1) was used in immunofluorescent staining to demonstrate the anatomical distribution of receptor protein expression. Using RT‐PCR for the CRF1 and CRF2 genes, both receptor gene transcripts were found in RNA isolated from crude colonic samples. CRF1 was found in the goblet and stem cells of the colonic crypts and in scattered cells of the surface epithelium and the lamina propria of the proximal colonic mucosa. In addition, double staining against neuron‐specific antigens revealed CRF1 expression in the myenteric and submucosal nervous plexus. CRF2 expression was localized mainly in the luminal surface of the crypts and in blood vessels of the submucosal layer. These results demonstrate expression of both CRF receptor types in the rat colon and support a role for their involvement in regulating peripheral effects of CRF ligands.

Amelioration of relapsing experimental autoimmune encephalomyelitis with altered myelin basic protein peptides involves different cellular mechanisms

T-cells specific for a region of human myelin basic protein, amino acids 87-99 (hMBP87-99), have been implicated in the development of multiple sclerosis (MS) a demyelinating disease of the central nervous system. Administration of soluble altered peptide ligand (APL), made by substituting native residues with alanine at either positions 91(91K > A or A91) or 97 (97R > A or A97) in the hMBP87-99 peptide, blocked the development of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE), in the SJL mouse. The non-encephalitogenic APL A91, appears to induce cytokine shifts from Th1 to Th2 in the target T-cells, whereas the encephalitogenic superagonist APL A97 causes deletion of the MBP87-99 responsive cells. Thus, single amino acid changes at different positions in the same peptide epitope can lead to APL capable of controlling auto-immune disease by different mechanisms.

A novel water-soluble selective CRF1 receptor antagonist, NBI 35965, blunts stress-induced visceral hyperalgesia and colonic motor function in rats.

The stress response involves the activation of two corticotropin-releasing factor (CRF) receptor subtypes. We investigated the role of CRF1 in stress-related visceral responses. A novel water-soluble tricyclic CRF1 antagonist, NBI 35965 was developed that displayed a high affinity for CRF1 (Ki approximately 4 nM) while having no binding affinity to CRF2. This antagonist also inhibited the stimulation of cAMP induced by sauvagine in CRF1 transfected cells. NBI 35965 administered per orally (p.o.) in rats (1, 3, 10 or 30 mg/kg) inhibited dose-dependently [125I]sauvagine binding selectively at brain sites of CRF1 distribution as shown by ex vivo receptor autoradiography. At the highest doses, NBI 35965 completely prevented [125I]sauvagine labeling in the cortex. NBI 35965 (10 mg/kg) administered p.o. or subcutaneously (s.c.) 1 h before intravenous CRF completely blocked the 81% shortening of distal colonic transit time induced by CRF. NBI 35965 (20 mg/kg s.c.) significantly reduced the defecation in response to water avoidance stress but not that induced by s.c. carbachol. In adult male Long-Evans rats that had undergone maternal separation, acute water avoidance stress significantly increased the visceromotor response to colorectal distention (20-80 mmHg) by 42+/-19% compared with the response before stress. Stress-induced visceral hyperalgesia was abolished by NBI 35965 (20 mg/kg, s.c.). The data show that NBI 35965 is a novel water-soluble selective CRF1 antagonist with bioavailability to the brain upon peripheral administration and that CRF1 receptor signaling pathways are involved in water avoidance stress-induced hyperalgesia to colorectal distention and stimulation of colonic transit.

CLONING OF A CDNA ENCODING A NOVEL INTERLEUKIN-1 RECEPTOR RELATED PROTEIN (IL1R-RP2)

We have identified and isolated both the rat and human cDNAs for a novel putative receptor related to the interleukin-1 type 1 receptor. We have named this protein interleukin 1 receptor related protein two (IL 1R-rp2). The rat cDNA for IL1R-rp2 was first identified using oligonucleotides of degenerate sequence in a polymerase chain reaction (PCR) paradigm with rat brain mRNA as the template. The protein encoded by both of these cDNAs are 561 amino acids long and exhibit 42% and 26% overall identity with the interleukin-1 type 1 and type 2 receptors, respectively. RNase protection assays from rat tissues revealed a predominant expression for IL 1R-rp2 in the lung and epididymis with lower levels detected in the testis and cerebral cortex. By in situ hybridization we were able to determine that the expression in rat brain appeared to be non-neuronal and associated with the cerebral vasculature. When expressed transiently in COS-7 cells the receptor was incapable of high affinity binding to either [125I]-recombinant human IL 1 alpha or [125I]-recombinant human IL 1 beta. Together, these data demonstrate the existence of a novel protein that is related to the interleukin-1 receptor but does not bind IL-1 by itself.

Differential profile of CRF receptor distribution in the rat stomach and duodenum assessed by newly developed CRF receptor antibodies

Peripheral corticotropin‐releasing factor (CRF) receptor ligands inhibit gastric acid secretion and emptying while stimulating gastric mucosal blood flow in rats. Endogenous CRF ligands are expressed in the upper gastrointestinal (GI) tissues pointing to local expression of CRF receptors. We mapped the distribution of CRF receptor type 1 (CRF1) and 2 (CRF2) in the rat upper GI. Polyclonal antisera directed against the C‐terminus of the CRF receptor protein were generated in rabbits and characterized by western blotting and immunofluorescence using CRF1‐ and CRF2‐transfected cell lines and in primary cultured neurons from rat brain cortex. A selective anti‐CRF1 antiserum (4467a‐CRF1) was identified and used in parallel with another antiserum recognizing both CRF1 and CRF2 (4392a‐CRF1&2) to immunostain gastric tissue sections. Antiserum 4467a‐CRF1 demonstrated specific immunostaining in a narrow zone in the upper oxyntic gland within the stomach corpus. Conversely, 4392a‐CRF1&2 labeled cells throughout the oxyntic gland and submucosal blood vessels. Pre‐absorption with the specific antigen peptide blocked immunostaining in all experiments. Doublestaining showed co‐localization of 4392a‐CRF1&2 but not 4467a‐CRF1 immunoreactivity with H/K‐ATPase and somatostatin immunostaining in parietal and endocrine cells of the oxyntic gland. No specific staining was observed in the antrum with either antisera, whereas only antiserum 4392a‐CRF1&2 showed modest immunoreactivity in the duodenal mucosa. Finally, co‐localization of CRF2 and urocortin immunoreactivity was found in the gastric glands. These results indicate that both CRF receptor subtypes are expressed in the rat upper GI tissues with a distinct pattern and regional differences suggesting differential function.

Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation.

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.

NBI-5788, an altered MBP83-99 peptide, induces a T-helper 2–like immune response in multiple sclerosis patients

We assessed the immune response induced in multiple sclerosis (MS) patients who had received NBI‐5788, an altered peptide ligand (APL) designed from an immunodominant region (83–99) of the neuroantigen myelin basic protein (MBP) (5, 10, or 20 mg subcutaneously weekly for 4 weeks). The mean frequency of NBI‐5788–responsive T cells (stimulation index > 3) in MS patients treated with the APL was 35.8 ± 12.8% (n = 7) compared with a mean frequency of 6.2 ± 1.3% (n = 7) for the untreated patients. The mean frequency of whole MBP–responsive T cells in MS patients treated with the APL was not significantly different from that of untreated patients (16.4 ± 5.7% vs 18.0 ± 6.3%, respectively). NBI‐5788–reactive T‐cell lines generated from NBI‐5788–treated patients exhibited an increased frequency of cross‐reactivity with MBP peptide 83–99 compared with NBI‐5788–reactive lines from control MS patients. Cytokine secretion by APL‐reactive T‐cell lines from NBI‐5788–treated MS patients was more frequently T‐helper 2–like compared with T‐cell lines from untreated MS patients. These results demonstrate that subcutaneous administration of a soluble APL based on the MBP peptide 83–99 in MS patients can induce an APL‐reactive immune response in which T lymphocytes cross‐reactive with the immunodominant neuroantigen MBP secrete anti‐inflammatory cytokines. The significant heterogeneity in immune response between individuals indicates the need for clinical laboratory correlation during the course of therapeutic trials. Ann Neurol 2000;48:758–765

A pharmacokinetic evaluation of five H1 antagonists after an oral and intravenous microdose to human subjects

AIMS To evaluate the pharmacokinetics (PK) of five H(1) receptor antagonists in human volunteers after a single oral and intravenous (i.v.) microdose (0.1 mg). METHODS Five H(1) receptor antagonists, namely NBI-1, NBI-2, NBI-3, NBI-4 and diphenhydramine, were administered to human volunteers as a single 0.1-mg oral and i.v. dose. Blood samples were collected up to 48 h, and the parent compound in the plasma extract was quantified by high-performance liquid chromatography and accelerator mass spectroscopy. RESULTS The median clearance (CL), apparent volume of distribution (V(d)) and apparent terminal elimination half-life (t(1/2)) of diphenhydramine after an i.v. microdose were 24.7 l h(-1), 302 l and 9.3 h, and the oral C(max) and AUC(0-infinity) were 0.195 ng ml(-1) and 1.52 ng h ml(-1), respectively. These data were consistent with previously published diphenhydramine data at 500 times the microdose. The rank order of oral bioavailability of the five compounds was as follows: NBI-2 > NBI-1 > NBI-3 > diphenhydramine > NBI-4, whereas the rank order for CL was NBI-4 > diphenhydramine > NBI-1 > NBI-3 > NBI-2. CONCLUSIONS Human microdosing provided estimates of clinical PK of four structurally related compounds, which were deemed useful for compound selection.