Generation of autonomously pathogenic neo-autoreactive Th1 cells during the development of the determinant spreading cascade in murine autoimmune encephalomyelitis

Chronic progression of autoimmune disease is accompanied by the acquisition of autoreactivity to new self-determinants. Recent evidence indicates that this process, commonly referred to as determinant spreading, may be pathogenic for chronicity. Our studies on experimental autoimmune encephalomyelitis (EAE), a murine model widely used in multiple sclerosis (MS) studies, have shown that determinant spreading develops as a predictable sequential cascade of neo-autoimmunity during progression to chronic disease. By 7-8 weeks after immunization of (SWR x SJL)F1 mice with the immunodominant myelin proteolipid protein determinant (PLP 139-151), splenocytes consistently respond to the immunodominant myelin basic protein determinant (MBP 87-99). In the present study, we directly address the pathogenicity of neo-autoimmunity resulting from endogenous self-priming during the course of disease. Our results indicate that T cells responding to the spreading MBP 87-99 determinant produce a proinflammatory cytokine profile consistent with type 1 helper T cells (Th1) cells. In addition, splenocytes activated to the spreading MBP 87-99 determinant consistently transfer acute EAE in naive recipients even when T cells reactive to the priming PLP 139-151 immunogen are eliminated by bromodeoxyuridine (BUdR)-mediated photolysis. Our data indicate that endogenous neo-autoantigen priming during chronic autoimmune disease generates type 1 helper T cells (Th1) cells that are autonomously pathogenic. These results provide further evidence supporting the view that determinant spreading is a pathogenic process that leads to chronic progression of autoimmune disease.     

PCR-based rapid detection of Mycobacterium tuberculosis in blood from immunocompetent patients with pulmonary tuberculosis

A PCR test based on insertion sequence IS1081 was developed to detect Mycobacterium tuberculosis complex organisms in the peripheral blood. The method was applied to blood samples from immunocompetent individuals with localized pulmonary tuberculosis. Seven of 16 (43.75%) blood samples were found to be positive for the circulating DNA copies of M. tuberculosis complex.     

Cigarette smoking increases sympathetic outflow in humans

OBJECTIVE: To describe a detailed operative procedure for type III laparoscopic radical hysterectomy with bilateral low paraaortic, subaortic and pelvic lymphadenectomy. STUDY DESIGN: Between January 1992 and December 1995, eight patients with cervical carcinoma IA2 or IB1 underwent laparoscopic radical hysterectomy at China Medical College Hospital, Taichung, Taiwan, R.O.C. The procedure of laparoscopic radical hysterectomy was separated into eight segmental steps. RESULTS: No major complications, including ureteral injury and lymphocyst formation, were noted in any case. Mean hospitalization was 6.5 days. The follow-up period ranged from 16 to 62 months. Only one case recurred, in the lung. CONCLUSION: Laparoscopic radical hysterectomy is a safe procedure. A complete pelvic and paraaortic lymphadenectomy and type III radical hysterectomy can be performed laparoscopically. This approach allows shorter hospitalization and carries less morbidity than the open type. Short-term follow-up (1.3-5.1 years) indicated a favorable prognosis.     

Inhibition of tissue repair by spironolactone: role of mineralocorticoids in fibrous tissue formation

The complete removal of the fluorescent cysteine derivative 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) from an intact protein has not been demonstrated even after extended treatment with a reducing agent. It has been suggested that this may be due to incomplete denaturation under the conditions employed. We were interested in investigating this phenomenon utilizing small peptides containing individual ABD-labeled cysteine residues. After incubating the fluorescent peptides in the presence of a reductant, it was shown that the ABD label could be completely removed from all of the cysteine-containing peptides investigated. Therefore, delabeling irreversibility is due to residual structure in proteins. Electrospray ionization mass spectrometry (ESI-MS) was used to determine the molecular mass of each peptide after removal of the ABD lavel. The ESI-MS data were consistent with the generation of a free sulfhydryl. The generation of the free sulfhydryl was further substantiated when a delabeled peptide was completely relabeled with ABD-F in the absence of reductant.     

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms

Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.     

Differential regulation of fast axonally transported proteins during the early response of rat retinal ganglion cells to axotomy

We have found that the early response of axotomized rat retinal ganglion cells is characterized by the differential regulation of a number of fast axonally transported proteins. The abundance of 23 radiolabeled fast transported proteins was analyzed at 2 and 5 days after axotomy using two-dimensional gel electrophoresis. Corresponding changes in retinal GAP-43 mRNA were measured using northern analysis. Within 2 days of injury, > 40% of the transported proteins analyzed, including GAP-43, showed increased labeling above control levels. Approximately 13% of transported proteins decreased below control levels, whereas the remainder did not change. Five days after axotomy, only GAP-43 and another fast transported protein, C3, continued to sustain measurable increased labeling above control levels; all previously elevated proteins appeared to have been down-regulated by this time, which corresponds to the onset of cell death. These differential changes were accompanied by parallel increases in GAP-43 mRNA. These results suggest that the molecular changes within rat retinal ganglion cells are differentially regulated within two stages subsequent to damage, initial regenerative growth followed by cell death.     

Adoptive immunotherapy with vaccine-primed lymph node cells secondarily activated with anti-CD3 and interleukin-2

PURPOSE: In preclinical studies, we have reported the ability to induce immune T cells in lymph nodes (LN) primed by in vivo vaccination with tumor cells admixed with a bacterial adjuvant. These LN cells can be activated and expanded ex vivo for the successful immunotherapy of established tumors. We have applied these methods to generate vaccine-primed LN in patients with advanced melanoma and renal cell cancer (RCC) for therapy. MATERIALS AND METHODS: Irradiated autologous tumor cells admixed with bacille Calmette-Guérin (BCG) were used to vaccinate patients. Seven days later, draining LN were removed for activation with anti-CD3 monoclonal antibody (mAb) followed by expansion in interleukin-2 (IL-2). Activated LN cells were administered intravenously (IV) with the concomitant administration of IL-2. RESULTS: A total of 23 patients were evaluated (11 melanoma and 12 RCC). Vaccine-primed LN were expanded ex vivo with a mean of 8.4 x 10(10) cells administered per patient. Among 20 patients assessed, 15 demonstrated minimal cytotoxicity of autologous tumor cells by the activated LN cells, with the remaining mediating nonspecific cytotoxicity. By contrast, a majority of the activated LN cells showed highly specific release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN-gamma) to autologous but not allogeneic tumor stimulation. This tumor-specific cytokine release was found to be major histocompatibility complex (MHC) class I-restricted, which indicates the involvement of CD8+ cells. Among 11 melanoma patients, one had a partial tumor response. Among 12 RCC patients, two had complete and two partial responses. A trend (P = .066) between the enhancement of delayed-type hypersensitivity (DTH) reactivity to autologous tumor after therapy and tumor regression was observed. CONCLUSION: Tumor vaccines can be used to induce immunologically specific T-cell responses against melanoma and RCC in draining LN. Anti-CD3/IL-2 activation of primed LN cells can be reliably performed for clinical therapy and appears to have activity in patients with metastatic RCC.