Charcot-Marie-Tooth neuropathy type 1B is associated with mutations of the myelin P0 gene.

P0, a major structural protein of peripheral myelin, is a homophilic adhesion molecule and maps to chromosome 1q22-q23, in the region of the locus for Charcot-Marie-Tooth neuropathy type 1B (CMT1B). We have investigated P0 as a candidate gene in two pedigrees with CMT1B and found point mutations which are completely linked with the disease (Z = 5.5, theta = 0). The mutations, glutamate substitution for lysine 96 or aspartate 90, are located in the extracellular domain, which plays a significant role in myelin membrane adhesion. Individuals with CMT1B are heterozygous for the normal allele and the mutant allele. Our results indicate that P0 is a gene responsible for CMT1B.

Evidence for clonal selection of gamma/delta T cells in response to a human pathogen.

T cells bearing gamma/delta antigen receptors comprise a resident population of intraepithelial lymphocytes in organs such as skin, gut, and lungs, where they are strategically located to contribute to the initial defense against infection. An important unsolved question about antigen-driven gamma/delta T cell responses regards the breadth of their T cell receptor (TCR) repertoire, since many specific epithelial compartments in mice display limited diversity. We have examined the diversity of TCR delta gene expression among human gamma/delta T cells from skin lesions induced by intradermal challenge with Mycobacterium leprae. We show that the vast majority of gamma/delta cells from M. leprae lesions use either V delta 1-J delta 1 or V delta 2-J delta 1 gene rearrangements and, within a given region of the lesion, display limited junctional diversity. This contrasts markedly with the extensive diversity of gamma/delta T cells from peripheral blood of these same individuals, as well as skin from normal donors. These results indicate that the gamma/delta response to M. leprae involves the selection of a limited number of clones from among a diverse repertoire, probably in response to specific mycobacterial and/or host antigens.

Defining protective responses to pathogens: cytokine profiles in leprosy lesions.

The immunological mechanisms required to engender resistance have been defined in few infectious diseases of man, and the role of specific cytokines is unclear. Leprosy presents clinically as a spectrum in which resistance correlates with cell-mediated immunity to the pathogen. To assess in situ cytokine patterns, messenger RNA extracted from leprosy skin biopsy specimens was amplified by the polymerase chain reaction with 14 cytokine-specific primers. In lesions of the resistant form of the disease, messenger RNAs coding for interleukin-2 and interferon-gamma were most evident. In contrast, messenger RNAs for interleukin-4, interleukin-5, and interleukin-10 predominated in the multibacillary form. Thus, resistance and susceptibility were correlated with distinct patterns of cytokine production.

Differential effects of cytolytic T cell subsets on intracellular infection.

In analyzing mechanisms of protection against intracellular infections, a series of human CD1-restricted T cell lines of two distinct phenotypes were derived. Both CD4(-)CD8(-) (double-negative) T cells and CD8(+) T cells efficiently lysed macrophages infected with Mycobacterium tuberculosis. The cytotoxicity of CD4(-)CD8(-) T cells was mediated by Fas-FasL interaction and had no effect on the viability of the mycobacteria. The CD8(+) T cells lysed infected macrophages by a Fas-independent, granule-dependent mechanism that resulted in killing of bacteria. These data indicate that two phenotypically distinct subsets of human cytolytic T lymphocytes use different mechanisms to kill infected cells and contribute in different ways to host defense against intracellular infection.

Ocular and cerebellar defects in zebrafish induced by overexpression of the LIM domains of the islet-3 LIM/homeodomain protein.

Islet-3 is an LIM/homeodomain protein that is expressed specifically in the eyes and the presumptive tectum in the central nervous system of zebrafish (Danio rerio) embryos. Overexpression of the protein (LIM(Isl-3)) consisting only of the Islet-3 LIM domains in embryos specifically prevented formation of the optic vesicles; caused abnormal termination of the expression of wnt1, engrailed2, and pax2 in the mesencephalic and metencephalic region between 14 hr and 20 hr postfertilization; and severely impaired morphogenetic movement in this region between 20 hr and 26 hr, which should normally lead to formation of the cerebellar primordium. Such defects were all rescued by simultaneous overexpression of Islet-3, suggesting that LIM(Isl-3) acted as a specific dominant-negative variant of Islet-3. These data, combined with the results of mosaic analyses, suggest that Islet-3 is activated by putative LIM-binding cofactors and functions to promote evagination of the optic vesicles and to maintain reciprocal interaction between the mesencephalon and the mesencephalic-metencephalic boundary essential for normal development of this region.

Functional repression of Islet-2 by disruption of complex with Ldb impairs peripheral axonal outgrowth in embryonic zebrafish.

Islet-2 is a LIM/homeodomain-type transcription factor of the Islet-1 family expressed in embryonic zebrafish. Two Islet-2 molecules bind to the LIM domain binding protein (Ldb) dimers. Overexpression of the LIM domains of Islet-2 or the LIM-interacting domain of Ldb proteins prevented binding of Islet-2 to Ldb proteins in vitro and caused similar in vivo defects in positioning, peripheral axonal outgrowth, and neurotransmitter expression by the Islet-2-positive primary sensory and motor neurons as the defects induced by injection of Islet-2-specific antisense morpholino oligonucleotide. These and other experiments, i.e., mosaic analysis, coexpression of full-length Islet-2, and overexpression of the chimeric LIM domains derived from two different Islet-1 family members, demonstrated that Islet-2 regulates neuronal differentiation by forming a complex with Ldb dimers and possibly with some other Islet-2-specific cofactors.

Interferon-gamma differentially regulates interleukin-12 and interleukin-10 production in leprosy.

The ability of monocytes to influence the nature of the T cell response to microbial pathogens is mediated in part by the release of cytokines. Of particular importance is the release of IL-12 and IL-10 by cells of the monocyte/macrophage lineage upon encountering the infectious agent. IL-12 promotes cell mediated immunity (CMI) to intracellular pathogens by augmenting T-helper type 1 responses, whereas IL-10 downregulates these responses. The ability of IFN-gamma to modulate the balance between IL-12 and IL-10 production was examined by studying leprosy as a model. In response to Mycobacterium leprae stimulation, IFN-gamma differentially regulated IL-12 and IL-10 production resulting in upregulation of IL-12 release and downregulation of IL-10 release. Furthermore, we determined that the mechanism by which IFN-gamma downregulates IL-10 was through the induction of IL-12. The data suggest a model of lymphocyte-monocyte interaction whereby the relative presence or absence of IFN-gamma in the local microenvironment is a key determinant of the type of monocyte cytokine response, and hence the degree of CMI in the host response to infection.

Cytokine patterns in the pathogenesis of human leishmaniasis.

The host response to infection appears to be regulated by specific patterns of local cytokine production. In the mouse, resistance to many pathogens including Leishmania is associated with a TH1 cytokine profile, IL-2 and IFN-gamma; whereas susceptibility to infection is associated with production of TH2 cytokines, IL-4, IL-5, and IL-10. To determine the cytokine patterns of the local immune response to Leishmania infection in humans, we used the polymerase chain reaction to compare cytokine mRNAs in biopsy specimens of American cutaneous leishmaniasis. In localized cutaneous leishmaniasis and the Montenegro delayed-type hypersensitivity reaction, type 1 cytokine mRNAs such as IL-2, IFN-gamma, and lymphotoxin were relatively predominant. In the chronic and destructive mucocutaneous form of leishmaniasis, there was a mixture of type 1 and type 2 cytokines, with a striking abundance of IL-4 mRNA in lesions. These results suggest that clinical course of infection with Leishmania braziliensis in man is associated with specific local patterns of cytokine production.

Cross-regulatory roles of interleukin (IL)-12 and IL-10 in atherosclerosis.

T cell cytokines are known to play a major role in determining protection and pathology in infectious disease. It has recently become clear that IL-12 is a key inducer of the type 1 T cell cytokine pattern characterized by production of IFN-gamma. Conversely, IL-10 down-regulates IL-12 production and type 1 cytokine responses. We have investigated whether IL-12 and IL-10 might be involved in a chronic inflammatory reaction, atherosclerosis. In atherosclerotic plaques, we found strong expression of IFN-gamma but not IL-4 mRNAs as compared to normal arteries. IL-12 p40 mRNA and IL-12 p70 protein were also found to be abundant in atherosclerotic plaques. IL-12 was induced in monocytes in vitro in response to highly oxidized LDL but not minimally modified LDL. The cross-regulatory role of IL-10 was indicated by the expression of IL-10 in some atherosclerotic lesions, and the demonstration that exogenous rIL-10 inhibited LDL-induced IL-12 release. These data suggest that the balance between IL-12 and IL-10 production contributes to the level of immune-mediated tissue injury in atherosclerotsis.

Human non-small cell lung cancer cells express a type 2 cytokine pattern.

In addition to infiltrating inflammatory cells, tumors also produce cytokines and growth factors that may alter tumor growth, tumor immunogenicity, and the host immune response. To characterize the expression profile of human non-small cell lung cancer (NSCLC)-derived cytokines, the mRNA expression of type 1 and type 2 cytokines in five human NSCLC lines was analyzed by reverse transcriptase-PCR. Expression of interleukin 5 (IL-5) and IL-10 was demonstrated in all tumor lines evaluated, whereas IL-4 was present in three of five lines and IL-13 was present in two of five lines. In contrast, none of the tumor lines expressed IL-2 and IFN-gamma. Type 2 cytokine protein production by NSCLC lines was confirmed by immunoprecipitation and cytokine specific ELISA. Tumor-derived IL-10 secretion was significantly augmented by exogenous recombinant cytokines including IL-4 and tumor necrosis factor-alpha. To evaluate whether fresh NSCLC nodules also express a type 2 cytokine pattern, the content of type 1 and type 2 cytokines in tissue homogenates from 13 fresh NSCLC nodules and normal lung surgical specimens was assessed. Human NSCLC nodules contain significantly more type 2 cytokines than does normal lung tissue when corrected for total protein concentration. To identify the cellular source of type 2 cytokine production in tumor nodules, immunohistology was performed on sections from 5 lung squamous cell carcinomas and 5 adenocarcinomas. All of the specimens revealed positive staining for type 2 cytokines within tumor cells. In summary, we report that human NSCLC cells produce type 2 cytokines both in situ and in vitro, which may play an active immunoregulatory role in the lung cancer microenvironment.

Isolation of neuronal plasma membranes from the crayfish Procamburus clarkii, with an aqueous two phase polymer system followed by sucrose density gradient centrifugation.

An aqueous two phase polymer system (Dextran-polyethyleneglycol system was developed for isolation of plasma membrane fraction from nerves of the crayfish, Procamburus clarkii. The polymer system effectively reduced both mitochondrial and endoplasmic reticulum marker enzyme activity from a crude membrane fraction. The similar enrichment of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was shown by the polymer system as well as by the sucrose density gradient centrifugation. The purified plasma membrane fraction (PM) was obtained using the polymer system followed by sucrose density gradient centrifugation. The PM fraction had a high specific activity of (Na + K+)-ATPase of up to 17 times that in the homogenate, with smaller contamination by mitochondria and endoplasmic reticulum enzyme activities than any other membrane fraction. Electron micrographs of the PM fraction also supported the above evidences. The protein recovered from the PM fraction amounted to 1.1% of the total protein in the homogenate. The specific activity of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in the membrane fractions was less increased than that of (Na+ + K+)-ATPase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis suggested that polypeptide chains of estimated molecular weight 115,000 and 31,000 were enriched in the plasma membranes of the crayfish nerves.

Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane.

Two major glycoproteins of bovine peripheral nerve myelin were isolated from the acid-insoluble residue of the myelin by a procedure involving delipidation with chloroform/methanol (2:1, v/v) and chromatography on Sephadex G-200 column with a buffer containing sodium dodecyl sulfate. The separation patterns of the proteins on the gel were affected considerably by the dodecyl sulfate concentration in the elution buffer. At above 2% dodecyl sulfate concentration in the elution buffer, the glycoproteins, could be separated clearly on the gel and were purified. The purified proteins, the BR protein (mol. wt. 28 000) and the PAS-II protein (mol. wt. 13 000), were homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2- terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose and sialic acids and the PAS-II protein contained glucosamine, mannose, galactose, fucose and glucose. Neither the BR protein nor the PAS-II were a glycosylated derivative of a basic protein of bovine peripheral nerve myelin, a deduction based on the results of amino acid analysis. The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits and guinea pigs, using dodecyl sulfate-polyacrylamide gel electrophoresis.

On basic proteins in bovine peripheral nerve myelin.

1. Myeline proteins in bovine peripheral nerve migrated as two main band-(BF and BR protein) and one faint middle band (BM protein) on sodium dodecyls sulfate-polyacrylamide gel electrophoresis. The relative mobility of these two main bands differed from those of myelin proteins in the central nervous system. 2. The acid extract of the myelin fraction from bovine peripheral nerve was separated into one main peak and two minor peaks on a Sephadex G-75 column. The major component of the second minor peak was the BM protein; the major component of the main peak was the BF protein. The BR protein was not extractable by acid solution. 3. Molecular weights of the BF, the BM and the BR protein were determined as around 13 000, 20 000 and 28 000, respectively, by sodium dodecylsulfate-polyacrylamide gel electrophoresis. 4. The amino acid composition of the BF protein was quite different from the encephalitogenic protein and the Folch-Lees type proteolipid protein in the central nervous system. However the BM protein showed similar amino acid composition to the encephalitogenic protein. 5. The tryptic peptide maps of the BF protein and of the encephalitogenic protein were quite different. The results suggested that the amino acid sequences of these two proteins are different and that they contain no common tryptophan-containing peptide.

Molecular cloning of cell adhesion molecule L1 from human nervous tissue: a comparison of the primary sequences of L1 molecules of different origin.

Complementary DNA for the human neural cell adhesion molecule L1 was cloned and sequenced: the deduced amino acid sequence consists of 1257 amino acid residues containing six repeats of the immunoglobulin C2 domain and five repeats of the fibronectin type III domain. The intracellular domain of human L1 is highly conserved as compared to mouse, but not identical to L1 cloned from human melanoma cells, suggesting the existence of alternative forms in the same species.

Cell-adhesion proteins of the immunoglobulin superfamily in the nervous system.

The unique groups of cell-adhesion proteins, such as IgSF, play essential parts in the formation and maintenance of the nervous system. Recent crystallographic studies have revealed a possible common structure of cell-adhesion proteins. The IgSF proteins are sub-grouped into simple, complex and mixed types. Accumulating evidence reveals the importance of cell-adhesion proteins in neural morphogenesis, maintenance and regeneration. They play key roles in neuronal migration, neurite outgrowth promotion, neurite fasciculation, pathfinding, target recognition, synaptogenesis and myelination. Mutations of cell-adhesion proteins result in neurological disease; for example, mutations of PO in hereditary neuropathy and mutations of L1 in hereditary hydrocephalus, MASA syndrome and spastic paraplegia type 1. Perspectives of the studies of neural cell-adhesion proteins are discussed.

Intravesical and intravenous therapy of human bladder cancer by the herpes vector G207.

G207, a conditionally replicating herpes vector, efficiently kills human bladder cancer cells in vitro. To evaluate the therapeutic potential of G207, we have established three in vivo models similar to the clinical situation. In vivo, G207 was intraneoplastically, intravesically, or intravenously inoculated in nude mice. Intraneoplastic inoculation into subcutaneous tumor caused significant tumor growth inhibition. Intravesical inoculation of G207 also caused decreased tumor growth in an orthotopic human bladder cancer model. Furthermore, multiple intravenous inoculation markedly inhibited subcutaneous tumor growth. These results suggest that intravesical therapy with G207 is effective for localized bladder tumor, especially for carcinoma in situ (CIS), and intravenous therapy with G207 is promising for invasive or metastasized bladder tumor.

The cytokine network in lesional and lesion-free psoriatic skin is characterized by a T-helper type 1 cell-mediated response.

As a psoriatic lesion develops at sites of previously uninvolved skin, cytokines and their subsequent induction of various adhesion molecules may play important pathophysiologic roles. To further define the cytokine network in psoriasis, biopsies were obtained from both lesional skin and lesion-free skin of individuals with psoriasis and compared to normal skin biopsies from control subjects. Each biopsy was analyzed using polymerase chain reaction for expression of cytokines and immunostaining to detect adhesion molecules. The results indicate that psoriatic lesions have a type 1 cytokine profile (i.e., interleukin[IL]-2, interferon[IFN]-gamma, and tumor necrosis factor[TNF]-alpha), without a significant component of type 2 cytokines (i.e., IL-4, IL-5, and IL-10) accompanied by aberrant expression of endothelial cell leukocyte adhesion molecule (ELAM)-1 and vascular cell adhesion molecule (VCAM)-1 on dermal endothelial cells, and ICAM-1 on epidermal keratinocytes. Four of five lesion-free biopsies from psoriatic patients had prominent cytokine mRNA expression compared with skin from normal donors (particularly TNF-alpha, IL-1 alpha, IL-1 beta, with lesser increases in IFN-gamma and granulocyte/macrophage colony-stimulating factor [GM-CSF]), which was accompanied by aberrant adhesion molecule expression in the same four samples. We conclude that a particular T-cell population producing type 1 cytokines accumulates in psoriatic lesions. In addition, clinically lesion-free skin is characterized by increased levels of various cytokine mRNAs, and aberrant adhesion molecule expression in both dermal and epidermal compartments.

Selective expansion of V delta 1 + T cells from leprosy skin lesions.

T cells bearing gamma delta T-cell receptors (TCRs) are prominent residents of murine epidermis and appear to be important participants in the immune response to infection in human skin. The Mitsuda reaction in leprosy, induced by intradermal challenge with Mycobacterium leprae, provides an opportunity to study the cellular events that mediate a form of delayed-type hypersensitivity (DTH) in skin. T cells bearing gamma delta TCRs comprise a significant proportion of the T-cell population in these DTH reactions. Presently we have generated T-cell lines from Mitsuda reactions in vitro and compared their TCR repertoire to that found in situ. gamma delta T cells comprised 20-40% of lines derived from these skin lesions, but < 10% of lines derived from the peripheral blood of the same individuals. Flow-cytometric analysis of variable (V) chain usage in T-cell lines derived from skin lesions indicated that V delta 1 was predominant. Evaluation of the TCR repertoire using PCR indicated that V delta 1-J delta 1 and V gamma 2-J gamma P gene rearrangements were prevalent. In comparison, V delta 2-J delta 1 gene rearrangements predominated in situ. Furthermore, nucleotide sequence analysis of the V-J junction of one T-cell line revealed limited genetic diversity of the gamma delta TCR. These findings suggest that the V delta 1 subpopulation of gamma delta T cells in Mitsuda skin reactions selectively outgrows from leprosy skin lesions in vitro. Such V delta 1 + T-cell lines should be useful for determining the relevant antigens and restriction elements in this response to a pathogen in skin.

Selective accumulation of T cells according to T-cell receptor V beta gene usage in skin cancer.

To investigate whether specific T-cell populations are overrepresented in tumor-infiltrating lymphocytes (TIL) in skin cancer, we determined the T-cell receptor (TCR) diversity in biopsy specimens of basal cell carcinoma and squamous cell carcinoma. Immunostaining of tissue sections indicated that the majority of T cells expressed alpha beta TCRs. To assess diversity of the TCR beta chain, RNA was isolated directly from the tumor specimens and peripheral blood mononuclear cells (PBMC) from the same patient, cDNA was synthesized, and variable (V) beta chain gene usage was determined by the polymerase chain reaction (PCR). In each basal cell (n = 11) and squamous cell (n = 7) carcinoma studied, several V beta families were overrepresented in TIL versus PBMC, in that they accounted for greater than 5% of the repertoire in TIL and were at least 2% higher in TIL than in PBMC. The predominant V beta gene segments overrepresented in TIL generally differed from individual to individual. Simultaneous comparison of the V beta repertoire of TIL to that of uninvolved skin and PBMC from the same individual revealed preferential expression of V beta families within the TIL in three of five basal cell and four of four squamous cell carcinomas. Again, the predominant V beta s differed from individual to individual. Comparison of the TCR repertoire in uninvolved skin versus PBMC did indicate that some V beta families were overexpressed in the resident T-cell compartment in skin, although the overrepresented families were not constant from individual to individual. These data indicate the selective concentration of T cells bearing specific alpha beta TCRs in the local immune response to basal cell and squamous cell carcinomas.

Molecular cloning of cDNA encoding the rat neural cell adhesion molecule L1. Two L1 isoforms in the cytoplasmic region are produced by differential splicing.

We have isolated and sequenced a full-length cDNA encoding the rat neural cell adhesion molecule L1. The deduced amino acid sequence as a whole shows high homology to mouse L1 sequence. In addition to this complete form of L1, we found an isoform, L1cs, which lacks four amino acid residues (RSLE) in the cytoplasmic domain and probably is derived from the same single L1 gene by tissue-specific alternative splicing. While L1 mRNA was predominantly expressed in the brain, L1cs mRNA was found exclusively in peripheral nervous tissue. Differential splicing in the highly conserved cytoplasmic domain may play an important role in modulating the function of L1 in different cells.