Human V␣24 NKT cells bearing an invariant V␣24J␣Q antigen receptor, the counterpart of murine V␣14 NKT cells, are activated by a specific ligand, ␣-GalCer, in a CD1d-dependent manner. Here, we demonstrate decreased numbers of circulating V␣24 NKT cells in patients with primary lung cancer compared to healthy volunteers. However, V␣24 NKT cells and DCs from lung cancer patients were functionally normal, even in the presence of tumor. Furthermore, levels of V␣24 NKT cells in surgically resected lung tissue appeared to be equivalent to those of V␣14 NKT cells in the mouse lung. Levels of V␣24 NKT cells in the tumor tissue itself were increased about 2.5 times. Administration of ␣-GalCer-pulsed DCs expanded V␣14 NKT cells in the lung more than 10 times, and the increased levels were sustained for 1 week. This may explain the previous finding that ␣-GalCer-pulsed DCs exerted strong antitumor activity in mouse lung tumor metastatic models. The potential use of ␣-GalCer-pulsed DCs for immunotherapy aimed at activating endogenous V␣24 NKT cells in the lung of cancer patients is discussed. © 2002 Wiley-Liss, Inc. Key words: V␣24 NKT cell; IFN-␥ primary lung cancer; ␣-galactosylceramide; single-cell sorting RT-PCRMurine V␣14 NKT cells are characterized by coexpression of NK1.1, an NK cell marker, and an invariant antigen receptor encoded by V␣14 and J␣281 gene segments. 1-3 V␣14 NKT cells recognize a glycolipid, ␣-GalCer, in a CD1d-depenent fashion. 4 The CD1d molecule has been well conserved throughout mammalian evolution and lacks allelic polymorphism. 5-7 After activation, V␣14 NKT cells inhibit tumor metastasis in certain experimental animal models. 8 -10 Furthermore, i.v. injection of ␣-GalCer-pulsed DCs expressed a potent antitumor effect in a B16 melanoma liver metastasis model, where metastasized tumor nodules were eradicated. 11 Human NKT cells bearing the invariant V␣24J␣Q receptor are considered to be the counterpart of murine V␣14 NKT cells and may recognize antigens similar to those of murine V␣14 NKT cells because of striking homology between human V␣24 and mouse V␣14 receptors, particularly in their complementarity-determining region 3. 2 Indeed, human V␣24 NKT cells were activated by ␣-GalCer in a CD1d-dependent fashion 5,12,13 and showed strong antitumor activity upon ␣-GalCer stimulation. 14,15 Human V␣24 NKT cells play crucial roles in various immune responses, including antitumor and autoimmune responses. 16 -19 These reports demonstrated selective reduction in V␣24 NKT cell numbers. Little is known, however, about the nature of V␣24 NKT cells in primary lung cancer patients.We investigated the function of V␣24 NKT cells in peripheral blood and lung of patients with lung cancer. The numbers of V␣24 NKT cells were reduced, whereas the function of freshly isolated V␣24 NKT cells appeared to be preserved in tumor-bearing patients. Furthermore, the ability to present ␣-GalCer by patient DCs was within a normal range. Administration of ␣-GalCer-pulsed DCs expanded V␣14 NKT cells in the lung more than 10 ti...

Patients with toxic epidermal necrolysis (TEN) have been known to have various complications. Though pulmonary complications are often observed, they usually show an acute form; however, chronic complications are quite rare and little is known about either their incidences or clinical manifestations. We herein report a 33-year-old man who presented with chronic pulmonary complications after a recovery from TEN. At the onset of TEN, he had severe respiratory failure and artificial ventilation was instituted. Despite being extubated successfully, respiratory failure reappeared 1 month later. A diagnosis of chronic bronchitis with severe obstructive ventilatory impairment and bronchiectasis was made and he was treated with steroids, bronchodilators and antibiotics, however, he died 1.5 years after the onset of TEN. There have been 13 reported cases of chronic pulmonary complications with TEN or Stevens-Johnson syndrome (SJS) in the English published work. Such cases are usually classified into chronic bronchitis/bronchiolitis with obstructive change (including bronchiolitis obliterans and bronchiolitis obliterans organizing pneumonia), respiratory tract obstruction and bronchiectasis. Approximately 40% of all such patients die while the surviving continue to suffer from these complications because no curative therapy yet exists. As a result, the prognosis seems to be poor. The relationship between TEN and these chronic pulmonary complications remains to be elucidated. Interestingly, our patient was asymptomatically anti-Ro/SS-A positive at the onset of TEN. In addition, eccrine gland involvement and an extremely high level of serum salivary amylase were observed at the onset of TEN, furthermore, Sjögren-like symptoms occurred after recovery from TEN. These findings suggested that the Sjögren-like autoimmune abnormalities induced by anti-Ro/SS-A correlated with the development of chronic pulmonary complications in our patient.

A unique lymphocyte lineage, the Vα14 NKT cells, expresses both NK1.1 and an invariant antigen receptor encoded by Vα14 and Jα281 gene segments. Vα14 NKT cells play crucial roles in various immune responses, including autoimmune diseases, allergic reactions and anti‐tumor immunity. Vα14 NKT cells were demonstrated to be essential for anti‐tumor effect of IL‐12 in vivo. Here, we report that adoptive transfer of IL‐12‐activated Vα14 NKT cells prevents hepatic metastasis of B16 melanoma. The injection of large amounts of IL‐2, IL‐4, and IFN‐γ, which are cytokines produced by activated Vα14 NKT cells, exhibited no significant inhibition of the metastasis of this melanoma. The cells prepared from the liver of IL‐12‐injected mice expressed a potent cytotoxic activity on B16 melanoma cells in vitro. Although the adoptive transfer of IL‐12‐activated Vα14 NKT cells prevents hepatic metastasis of B16 melanoma, activated NK cells from IL‐12‐injected RAG‐1−/− mice failed to inhibit the metastasis of this melanoma. Thus, the anti‐tumor effect of IL‐12 can be replaced by adoptive transfer of IL‐12‐activated Vα14 NKT cells but not by IL‐12‐activated NK cells, suggesting a minor role of NK cells for the IL‐12‐mediated anti‐tumor effect in this experimental system. Moreover, our studies have suggested the involvement of direct cytotoxic mechanisms rather than cytokine‐mediated immune responses at the effector phase of the Vα14 NKT cell‐mediated anti‐tumor activity. © 2001 Wiley‐Liss, Inc.

Transketolase, one of the enzymes in the nonoxidative branch of the pentose phosphate pathway, operates to shuttle ribose 5-phosphate and glycolytic intermediates together with transaldolase, and might be involved in the availability of ribose 5-phosphate, a precursor of nucleotide biosynthesis. The tkt and tal genes encoding transketolase and transaldolase, respectively, were cloned from the typical nucleotide- and nucleoside-producing organism Corynebacterium ammoniagenes by a PCR approach using oligonucleotide primers derived from conserved regions of each amino acid sequence from other organisms. Enzymatic and molecular analyses revealed that the two genes were clustered on the genome together with the glucose 6-phosphate dehydrogenase gene (zwf). The effect of transketolase modifications on the production of inosine and 5'-xanthylic acid was investigated in industrial strains of C. ammoniagenes. Multiple copies of plasmid-borne tkt caused about tenfold increases in transketolase activity and resulted in 10-20% decreased yields of products relative to the parents. In contrast, site-specific disruption of tkt enabled both producers to accumulate 10-30% more products concurrently with a complete loss of transketolase activity and the expected phenotype of shikimate auxotrophy. These results indicate that transketolase normally shunts ribose 5-phosphate back into glycolysis in these biosynthetic processes and interception of this shunt allows cells to redirect carbon flux through the oxidative pentose pathway from the intermediate towards the purine-nucleotide pathway.

A novel lymphocyte, NKT cells bearing an invariant V(alpha)14 antigen receptor, specifically recognizes alpha-galactosylceramide (alpha-GalCer) exclusively presented by mouse CD1d (mCD1d). However, the precise molecular interaction remains unclear. For the basis of functional analyses, a docking model of alpha-GalCer with the crystal structure of mCD1d was constructed. Possible residues involved in the alpha-GalCer--mCD1d interaction were found to be Arg79, Glu83 and Asp80 for carbohydrate recognition, and Asp153 for interaction with the amide group on the fatty acyl chain. The alpha-GalCer-presenting ability of various transfectants expressing mutant mCD1d was completely abrogated if a single amino acid mutation was induced at positions 79, 80, 83 or 153, suggesting that the polar amino acids above the F' pocket are crucial for alpha-GalCer presentation to activate V(alpha)14 NKT cells. The possibility that Glu83 is a contact site for the NKT cell receptor is also discussed.

NKT cells, a novel murine lymphoid lineage bearing an invariant T cell receptor encoded by Vα14 and Jα281 gene segments, recognize a specific ligand glycolipid, α‐galactosylceramide (α‐GalCer) in a CDld‐dependent manner. Recent research has revealed that activated Vα14 NKT cells have dramatic antitumor effects against a wide variety of tumor cell lines in vivo and in vitro. Here, we demonstrate strong in vivo antitumor effects brought about by treatment with α‐GalCer‐pulsed dendritic cells in comparison with in vitro‐activated Vα14 NKT cells. Furthermore, we show a significant expansion of endogenous Vα14 NKT cells in the lung following the administration of α‐GalCer‐pulsed dendritic cells. The feasibility of immunotherapy with α‐GalCer‐pulsed dendritic cells is discussed.

Five mutants were obtained, Y11, Y135, Y164, Y180 and Y61, capable of accumulating higher amounts of arachidonic acid (AA) than Mortierella alpina 1S-4, an industrial strain for the production of AA-rich triacylglycerol (TG). This is thought to be due to low or no activity of n-3 desaturation with conversion of AA to eicosapentaenoic acid, which functions at a cultural temperature below 20 degrees C. In small-scale cultivation under optimum conditions, Y11 and Y61 respectively accumulated 4.97 mg/ml and 4.11 mg/ml of AA, using a high concentration of glucose at 20 degrees C, compared with 3.74 mg/ml for M. alpina 1S-4. In a 5-1 jar fermentor, the AA content in Y11 and Y61 kept increasing during cultivation, with consumption of the glucose in the medium; and this reached 1.48 mg/ml and 1.77 mg/ml (118 mg/g, 120 mg/g of dry mycelia) at day 10, respectively, compared with 0.95 mg/ml (86 mg/g of dry mycelia) for M. alpina 1S-4. From the results of lipid analysis, the TG contents of Y11 and Y61 in the major lipids were significantly higher than that of M. alpina 1S-4; and the AA percentages in TG of Y11 and Y61 were also higher. Both Y11 and Y61 are potential producers of TG rich in AA.

A 57-year-old Japanese woman who suffered from alopecia areata associated with myasthenia gravis (MG) and thymoma responded well to thymectomy and high doses of glucocorticosteroid administration. Several treatments for alopecia areata including administration of systemic prednisolone were attempted, but loss of hair on the scalp progressed. After thymectomy and high level glucocorticosteroid administration for MG, the lesions on the scalp improved within four weeks. Consequently, we suggest that this thymectomy and high level glucocorticosteroid administration assisted in improving the immune dysfunction causing the alopecic lesions in this patient.