Intestinal Coccidiosis in a Patient with Alpha-chain Disease

During an ultrastructural study of small-intestinal mucosa from a patient suffering from alpha-chain disease organisms were identified within the epithelial cytoplasm which showed the fine structural features of the coccidian group. Though coccidiosis is well recognized as causing a diarrhoeal and often lethal illness in animals it has been neglected as a cause of disease in man. Thus this finding may be significant and warrants further investigation into its possible role in the pathogenesis of alpha-chain disease.     

Lymphokine-activated and natural killer cell activity in human intestinal mucosa

The activity of natural effector (NE) cells was studied in lamina propria lymphocytes (LPL) obtained from 61 histologically normal specimens of human intestine, which included 45 resected for colon carcinoma and 16 resected for nonmalignant conditions. The mean spontaneous natural killer (NK) cell activity in LPL (1.7 X 10(2) cytotoxic units (C.U.)/10(5) cells) was very low in contrast to that found in peripheral blood mononuclear cells (PBMC) (38.5 X 10(2) C.U./10(5) cells). Significant NK activity was detected in only 16 (47%) of the tissues resected for carcinoma, and in five (38%) of those removed for nonmalignant conditions. Exposure to human leucocyte interferon resulted in only minimal increases in cytotoxicity for K562 target cells. Consistent with these findings, large granular lymphocytes represented less than 0.5% of freshly isolated LPL. Cultures of LPL from both carcinoma and nonmalignant conditions in MLA144-conditioned medium (CM), a source of interleukin 2 (IL 2), generated marked increases in cytotoxicity to levels comparable with or exceeding those found in PBMC. (Mean cytotoxicities were 90.4 X 10(2) and 49 X 10(2) C.U./10(5) cells, respectively.) Cytotoxicity induced by culture in MLA144-CM could be blocked by pretreatment of LPL with the monoclonal antibody anti-Tac directed against the IL 2 receptor. In addition, LPL cultured in recombinant human IL 2 were induced to levels of cytotoxicity that were similar to those induced by MLA144-CM. These data indicate that IL 2 is the factor in MLA144-CM responsible for generating lymphokine-activated killer (LAK) cells in LPL. The IL 2-activated LPL killer cells were OKT11+, OKT3-, Leu-7-, Leu-11b-, as determined by antibody and complement-mediated lysis, and the precursor cells in the lamina propria necessary for generation of killer cells by IL 2 were also OKT11+, OKT3-, Leu-7-, Leu-11b-. These studies indicate that LAK cells may be an important potential source of nonspecific cytotoxicity in the intestinal mucosa.     

Cloning and characterisation of the Schizosaccharomyces pombe rad8 gene, a member of the SNF2 helicase family.

The Schizosaccharomyces pombe rad8 mutant is sensitive to both UV and gamma irradiation. We have cloned the rad8 gene by complementation of the UV sensitivity of a rad8.190 mutant strain. The gene comprises an open reading frame of 3.4 kb which does not contain any introns and is capable of encoding a 1133 amino acid protein of 129 kDa. Deletion of the gene indicates that it is not essential for cell viability. Recognisable motifs are present for a nuclear localisation signal, a RING finger and helicase domains. The predicted protein is a member of the SNF2 subfamily of proteins and shows particular homology to the Saccharomyces cerevisiae RAD5 protein. Double mutant analysis demonstrated that the rad8 mutant is not epistatic to mutants in the excision repair pathway (rad13) or checkpoint pathway (rad9). Analysis of radiation sensitivity though the cell cycle indicates that, unlike most other rad mutants, rad8 is most sensitive to irradiation during the G1/S period.     

Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.     

Depleted mucosal antioxidant defences in inflammatory bowel disease

Experimental approaches designed to define the role of reactive oxygen and nitrogen species generated by inflammatory cells in the tissue injury seen in inflammatory bowel disease rarely consider the chemical antioxidant defences against such increased oxidant stress in the mucosa. In this investigation, we have analysed components of the aqueous and lipid phase antioxidant mucosal defences by measuring the total peroxyl radical scavenging capacity and the levels of urate, glutathione, -tocopherol, and ubiquinol-10 in paired noninflamed and inflamed mucosal biopsies from inflammatory bowel disease patients. Compared to paired noninflamed mucosa, decreases were observed in inflamed mucosa for total peroxyl radical scavenging capacity (55%, p = 0.0031), urate [Crohn's disease (CD), 62.2%, p = 0.066; ulcerative colitis (UC), 47.3%, p = 0.031], glutathione (UC, 59%, 7/8 patients, ns), total glutathione (UC 65.2%, 6/8 patients, ns), ubiquinol-10 (CD, 75.7%, p = 0.03; UC, 90.5%, p = 0.005). The mean -tocopherol content was unchanged. These observations support our earlier findings of decreased reduced and total ascorbic acid in inflamed IBD mucosa and demonstrate that the loss of chemical antioxidant defences affects almost all the major components. The decreased antioxidant defences may severely compromise the inflamed mucosa, rendering it more susceptible to oxidative tissue damage, hindering recovery of the mucosa and return of epithelial cell layer integrity. The loss of chemical antioxidant components provides a strong rationale for developing novel antioxidant therapies for the treatment of inflammatory bowel disease.     

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.     

Leukotactin-1-induced ERK activation is mediated via Gi/Go protein/PLC/PKC delta/Ras cascades in HOS cells

Recently cloned leukotactin-1 (Lkn-1) that belongs to CC chemokine family has not been characterized. To understand the intracellular events following Lkn-1 binding to CCR1, we investigated the activities of signaling molecules in response to Lkn-1 in human osteogenic sarcoma cells expressing CCR1. Lkn-1-stimulated cells showed elevated phosphorylation of extracellular signal-related kinases (ERK1/2) with a distinct time course. ERK activation was peaked in 30 min and 12 h showing biphasic activation of ERK. Pertussis toxin, an inhibitor of G(i)/G(o) protein, and phospholipase C (PLC) inhibitor blocked Lkn-1-induced activation of ERK. Protein kinase C delta (PKC delta) specific inhibitor rottlerin inhibited ERK activation in Lkn-1-stimulated cells. The activities of PLC and PKC delta were also enhanced by Lkn-1 stimulation. Dominant negative Ras inhibited activation of ERK. Immediate early response genes such as c-fos and c-myc were induced by Lkn-1 stimulation. Lkn-1 affected the cell cycle progression by cyclin D(3) induction. These results suggest that Lkn-1 activates the ERK pathway by transducing the signal through G(i)/G(o) protein, PLC, PKC delta and Ras, and it may play a role for cell proliferation, differentiation, and regulation of gene expression for other cellular processes.