Experience of Quatro-Therapy With Everolimus to Minimize Calcineurin Inhibitor for Kidney Transplant Recipients.

BACKGROUND: This study was divided into three phases, on the occasion of the introduction of everolimus (EVR) in our hospital. METHODS: In the first phase, a study group of six maintenance patients (three living related donors, three deceased donors) who had a history of malignant disease with less than 500 mg/day of proteinuria were enrolled; a high serum creatinine and upper limit of duration after kidney transplant operation was not considered. EVR was discontinued in four of the six patients because of side effects or worsening renal function. The second phase comprised a study group of 12 maintenance patients (12 living related donors) who were more than 5 years after kidney transplant operation with serum creatinine <3 ng/mL and proteinuria <500 mg/day. In two patients, EVR was discontinued because of a skin rash or general fatigue, but EVR was continued in 10 cases. Calcineurin inhibitor (CNI) dosage was reduced and renal function improved, and mean estimated glomerular filtration rate recovered from 42.3 mL/min to 44.8 mL/min, with no rejections occurring. In the third phase, a study group of eight de novo transplant patients who were 2 to 3 weeks after transplant operation were examined. In one case, EVR was discontinued because of proteinuria but was restarted with a stepwise increasing method after 4 months and was continued without any side effects. RESULTS: Our study indicates that EVR was a useful drug for the maintenance of kidney transplant recipients for the optimal patients. CONCLUSIONS: In de novo cases, EVR plus a high dose of mizoribine and low CNI protocol was a useful regimen without serious adverse effects.

An alignment method for mammographic X-ray spectroscopy under clinical conditions.

This paper describes an alignment method for mammographic X-ray spectroscopy under clinical conditions. A pinhole, a fluorescent screen, a laser device and the case for a detector are used for alignment of the focal spot, a collimator and a detector. The method determines the line between the focal spot and the point of interest in an X-ray field radiographically. The method allows alignment for both central axis and off-axis directions.

Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro.

The pathogenesis of pseudomonal keratitis was investigated by focusing on induction and activation of matrix metalloproteinases (MMPs) by pseudomonal virulence factors and proinflammatory cytokines. Corneal lesions and MMP induction in vivo were evaluated in rabbit corneas infected with a clinical isolate of Pseudomonas aeruginosa. Effects of pseudomonal virulence factors [elastase, alkaline protease, exotoxin A and lipopolysaccharide (LPS)], tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta on MMP induction and activation were further examined in vitro in rabbit corneal fibroblasts (RCF) and human fibrosarcoma (HT1080) cells using reverse transcriptase-polymerase chain reaction (RT-PCR), zymography and immunoblotting. Corneal ulcers with typical ring abscesses were observed 12-24 h after infection, and MMPs, particularly MMP-9, were upregulated in infected corneas. Pseudomonal elastase caused the most extensive damage to both cell types. RCF treated with pseudomonal exoproteases or LPS expressed and secreted MMP-9. Exotoxin A had no effect on MMP expression. Both IL-1beta and TNF-alpha augmented MMP-9 expression in HT1080 cells. Pseudomonal elastase proteolytically activated MMP-2 and MMP-9 released from the cells. In conclusion, corneal destruction seen with P. aeruginosa infections may result from enhanced expression of MMPs by corneal stromal cells stimulated with pseudomonal exoproteases and proinflammatory cytokines and the proteolytic activation of MMPs by pseudomonal elastase.

Stimulative effect of cadmium on prostaglandin E2 production in primary mouse osteoblastic cells.

We have reported that cadmium (Cd) stimulates bone resorption via prostaglandin E2 (PGE2), which is mainly produced in osteoblasts. Prostaglandin (PGs) is regulated by arachidonic acid (AA) release by phospholipase A2 (PLA2) and its conversion to PGs by cyclooxygenase (COX). In the present study, we investigated the possibility that Cd-induced PGE2 synthesis was mediated through PLA2 or COX or both using primary mouse osteoblastic cells in serum-free medium. Cd at 1 microM and above stimulated 14C-AA release from 14C-AA-prelabeled osteoblastic cells. PLA2 activity of cytosolic fraction in Cd-treated cells preferentially hydrolyzed AA at the Sn2 position of phospholipids and was inhibited by arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2 (cPLA2). Cd at 1 microM and above increased cPLA2 activity and the level of constitutive cPLA2 mRNA. Secretory PLA2 mRNA was not detected. On the other hand, Cd at 1 microM and above stimulated PGE2 production and its production was inhibited by an inhibitor of COX-2 (NS-398). Cd at 1 microM and above markedly stimulated COX-2 mRNA expression and slightly increased the level of COX-1 mRNA. An inhibitor of COX-1 (varelylsalicylic acid) did not affect Cd-induced PGE2 production. In addition, Cd-induced PGE2 synthesis was inhibited by AA-COCF3, On the other hand, IL-1 alpha, an inducer of COX-2, did not stimulated PGE2 production in present culture system. When IL-1 alpha- or Cd-treated cells were incubated with AA for 10 minutes, IL-1 alpha-treated cells as well as Cd-treated ones caused an increase in PGE2 production. This suggests that the mechanism of Cd-induced PGE2 production is different from that of IL-1 alpha, which may require an activation of cPLA2. From these results, it was found that Cd by itself stimulated PGE2 production by two successive steps that Cd increased cPLA2 activity and then COX-2 induction.

Salivary gland MALT lymphoma associated with Helicobacter pylori infection in a patient with Sjögren's Syndrome.

We report a case of salivary gland MALT lymphoma in Sjögren's syndrome associated with localized H. pylori infection. A 76-year-old woman had a history of bilateral cheek masses for two years. Histologically, the parotid glands were invaded by numerous centrocyte-like cells to form lymphoepithelial lesions. The tumor cells showed immunohistological differentiation into B cells. Southern blotting demonstrated immunoglobulin gene rearrangement. These results indicated that the tumors were MALT lymphoma. H. pylori, as assessed by the urease test (CLO test; BML Ltd., Tokyo, Japan), was positive in the tumor specimen. After wide local excision of the tumors followed by radio therapy and oral administration of antibiotics and proton pump inhibitor, no evidence of recurrence was found during the 24-months of follow up. H. pylori infection in the salivary gland is rare, although the source of infection and transmission of H. pylori organisms has been thought to be the oral cavity. We discussed the association between H. pylori infection and salivary gland MALT lymphoma. The microorganism may play a role as an additional antigenic stimulus for the development of salivary gland MALT lymphoma as well as for the development of gastric MALT lymphoma. This means that H. pylori can play a role in lymphoma progression as booster of B cell lymphoproliferation.

Cell density-dependent regulation of proteoglycan synthesis by transforming growth factor-beta(1) in cultured bovine aortic endothelial cells.

The regulation of vascular endothelial cell behavior during angiogenesis and in disease by transforming growth factor-beta(1) (TGF-beta(1)) is complex, but it clearly involves growth factor-induced changes in extracellular matrix synthesis. Proteoglycans (PGs) synthesized by endothelial cells contribute to the formation of the vascular extracellular matrix and also influence cellular proliferation and migration. Since the effects of TGF-beta(1) on vascular smooth muscle cell growth are dependent on cell density, it is possible that TGF-beta(1) also directs different patterns of PG synthesis in endothelial cells at different cell densities. In the present study, dense and sparse cultures of bovine aortic endothelial cells were metabolically labeled with [(3)H]glucosamine, [(35)S]sulfate, or (35)S-labeled amino acids in the presence of TGF-beta(1). The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain, heparitinase, or chondroitin ABC lyase. These experiments indicate that the effect of TGF-beta(1) on vascular endothelial cell PG synthesis is dependent on cell density. Specifically, TGF-beta(1) induced an accumulation of small chondroitin/dermatan sulfate PGs (CS/DSPGs) with core proteins of approximately 50 kDa in the medium of both dense and sparse cultures, but a cell layer-associated heparan sulfate PG with a core protein size of approximately 400 kDa accumulated only in dense cultures. Moreover, only in the dense cell cultures did TGF-beta(1) cause CS/DSPG hydrodynamic size to increase, which was due to the synthesis of CS/DSPGs with longer glycosaminoglycan chains. The heparan sulfate PG and CS/DSPG core proteins were identified as perlecan and biglycan, respectively, by Western blot analysis. The present data suggest that TGF-beta(1) promotes the synthesis of both perlecan and biglycan when endothelial cell density is high, whereas only biglycan synthesis is stimulated when the cell density is low. Furthermore, glycosaminoglycan chains are elongated only in biglycan synthesized by the cells at a high cell density.

A case of calcinosis universalis successfully treated with low-dose warfarin.

A 27-year-old male patient with calcinosis universalis resulting from dermatomyositis was successfully treated with low-dose warfarin. On his trunk and extremities, there were many subcutaneous calcified nodules, and knee flexion was difficult. After oral warfarin therapy for three years, the calcified nodules became smaller, and the knee mobility improved. His serum vitamin K level was abnormally high, decreased just after starting warfarin therapy, and then remained within the normal range. Since vitamin K has been known to play an important role in the Ca2+ binding process in bones or tissues, we suggest that this therapy is effective in reducing subcutaneous calcification through the vitamin K cycle.

Lecithin-bound superoxide dismutase in the prevention of neutrophil-induced damage of corneal tissue.

PURPOSE: To evaluate the effects of a lipophilic analog of superoxide dismutase (SOD) in the prevention of polymorphonuclear leukocyte (PMN)-induced damage to corneal epithelial cells in vitro and in bacterial corneal ulcers in vivo. METHODS: Immortalized human corneal epithelial cells (T-HCEC) were cocultured with human PMNs activated with N-formyl-methionyl-leucyl-phenylalanine for 18 hours, after which lactate dehydrogenase (LDH) activity of the supernatant was measured as a marker of cellular damage. The inhibitory effects of lecithin-bound SOD (PC-SOD) and unmodified SOD, as well as PMNs pretreated with anti-CD 18 monoclonal antibody, were compared with untreated control. The retention of each drug on the ocular surface of healthy volunteers was measured by flow cytometry using brush cytology samples. The protective effects of a 0.1% solution of PC-SOD on Pseudomonas aeruginosa corneal infection in guinea pigs were assessed by inflammatory grading scores and histology. RESULTS: Both PC-SOD and SOD effectively suppressed PMN-induced LDH release in T-HCEC in a dose-dependent manner. LDH release was also attenuated when PMNs were pretreated with anti-CD 18 antibodies, suggesting that adhesion molecules were involved in the process. Brush cytology of conjunctival samples showed that PC-SOD was retained longer on the ocular surface compared with unmodified SOD. PC-SOD significantly prevented excessive tissue damage by infiltrating PMNs in P. aeruginosa corneal infection, whereas in control eyes, perforation of the cornea occurred by 6 days. CONCLUSIONS: PC-SOD was effective in attenuating PMN-related tissue damage to corneal tissue both in vitro and in P. aeruginosa infection in guinea pigs.

[Hypertrophic cardiomyopathy showing no 123I-BMIPP myocardial accumulation with type I CD36 deficiency].

A 57 years old male consulted our hospital in complaining chest oppression and short of breath. Familial and dilated phase hypertrophic cardiomyopathy (HCM) was detected by ECG, echocardiography, left ventriculography and left ventricular endomyocardial biopsy. 201T1 SPECT showed regional increased accumulation in the ventricular septum, however, no myocardial accumulation of 123I-beta-methyl-p-iodophenylpentadecanoic acid (123I-BMIPP) was observed. We analyzed CD36 in this patient, and found he had type 1 CD36 deficiency. Myocardial uptake of long-chain fatty acids occurs via a specific transporter, which is homologous with human CD36. We hypothesize that CD36 deficiency, especially type 1 CD36 deficiency, might be one factor of no myocardial 123I-BMIPP uptake.

Activation of human matrix metalloproteinases by various bacterial proteinases.

Matrix metalloproteinases (MMPs) are zinc-containing proteinases that participate in tissue remodeling under physiological and pathological conditions. To test the involvement of bacterial proteinases in tissue injury during bacterial infections, we investigated the activation potential of various bacterial proteinases against precursors of MMPs (proMMPs) purified from human neutrophils (proMMP-8 and -9) and from human fibrosarcoma cells (proMMP-1). Each proMMP was subjected to treatment with a series of bacterial proteinases at molar ratios of 0.01-0.1 (bacterial proteinase to proMMP), and activities of MMPs generated were determined. Among six different bacterial proteinases, thermolysin family enzymes (family M4) such as Pseudomonas aeruginosa elastase, Vibrio cholerae proteinase, and thermolysin strongly activated all three proMMPs via limited proteolysis to generate active forms of the MMPs. N-terminal sequence analysis of the active MMPs revealed that cleavage occurred at the Val82-Leu83 and Thr90-Phe91 bonds of proMMP-1 and proMMP-9, respectively, which are located near the N terminus of the catalytic domain of MMPs. In contrast, Serratia 56-kDa proteinase and Pseudomonas alkaline proteinase, both of which are classified as members of the serralysin subfamily of zinc metalloproteinases (family M10), and Serratia 73-kDa thiol proteinase did not evidence proteolytic processing or activation of proMMP-1, -8, and -9 under these experimental conditions. These results indicate that bacterial proteinases may play an important role in tissue destruction and disintegration of extracellular matrix at the site of infections.

Sensitive response of cultured vascular smooth-muscle cells to cadmium cytotoxicity: comparison with cultured vascular endothelial cells and kidney epithelial LLC-PK1 cells.

Response to cadmium cytotoxicity of cultured bovine aortic smooth-muscle cells was compared with that of cultured bovine aortic endothelial cells and porcine kidney epithelial LLC-PK1 cells. The cell damage was evaluated by morphology and the lactate dehydrogenase leakage assay. It was found that vascular smooth-muscle cells are markedly sensitive to cadmium cytotoxicity. The accumulation of intracellular cadmium was much higher but that of metallothionein was much less in vascular smooth-muscle cells than in LLC-PK1 cells; vascular endothelial cells were in between vascular smooth-muscle cells and LLC-PK1 cells. The content of reduced glutathione was slightly increased by cadmium in all three cell types. The present data suggest that a much lower inducibility of metallothionein with a high accumulation of intracellular cadmium in vascular smooth-muscle cells resulted in a marked sensitivity of the cells to cadmium cytotoxicity. Vascular smooth-muscle cells may be one of the critical target of cadmium toxicity.

Vascular smooth muscle cells in culture are highly sensitive to cadmium cytotoxicity without species-related differences: comparison with Chang liver cells.

Human, rabbit and rat aortic smooth muscle cells (hSMC, rSMC and A10 cells, respectively) were cultured with cadmium chloride and compared with human Chang liver cells to characterize the response of vascular smooth muscle cells to the metal. It was revealed that all tested vascular smooth muscle cells were markedly more sensitive to cadmium cytotoxicity than Chang liver cells. Cadmium accumulated more markedly in vascular smooth muscle cells than in Chang liver cells. After exposure to cadmium, metallothionein was induced in a concentration-dependent manner in Chang liver cells, it was constitutively high in hSMC, sensitively induced in rSMC and was constitutively low and induced within narrow limits in A10 cells. The intracellular content of reduced glutathione was greater and significantly enhanced by cadmium only in A10 cells. The present data suggest that vascular smooth muscle cells are, in general, sensitive to cadmium cytotoxicity without any species-related differences, mainly due to a higher accumulation of the metal within cells.

[Detection of loss of heterozygosity by microsatellite probe and DNA content analysis].

We examined replication error (RER) and loss of heterozygosity (LOH) in the region of microsatellites in 60 cases of resected lung cancer. We used microsatellite probes for the short arm of the 2nd chromosome (D2S123, D2S136), the short arm of the 3rd chromosome (D3S1067), and the short arm of the 17th chromosome (TP53). According to stage, the frequency of LOH was 25% in stage I, 33% in stage II, 44% in stage IIIA, 11% in stage III B, and 63% in stage IV. According to histological classification, the frequency of LOH was 41% for squamous cell carcinoma, 24% for adenocarcinoma, and 100% for small cell carcinoma. According to microsatellite probe results, the frequency of LOH was 6.7% for D2S123, 5.0% for D2S136, 16.7% for D3S1067, and 18.3% for TP53. Two of the 60 cases showed RER. One case was stage I squamous cell carcinoma, and the other was stage IV adenocarcinoma. Except for stage III B,LOH in the microsatellite region increases with the stage. LOH is often detected in the order of small cell carcinoma, squamous cell carcinoma, and adenocarcinoma. According to the chromosome number, LOH is detected more often in the 3rd and 17th chromosomes than in the 2nd chromosome. In 20 cases with LOH, only two showed DNA diploidy. Compared to LOH of the microsatellite region, DNA content analysis by flow cytometry has accuracy problems.

[The result of reoperation for lung cancer].

Twenty four patients with recurrent or multiple lung cancer were reoperated in our center. Five-year survival rate was 20% for 11 patients with recurrent, while was 25% for 13 patients with multiple after reoperation. The patients with limited operation had well survival and there was no significant difference in procedure. However all four patients with N2 had poor prognosis. Seven patients (29%) had the post reoperative complication in pulmonary system. All of them had the impairment of pulmonary function (FEV1.0% was less than 50%) or more than 75% perfusion ratio, measured with pulmonary perfusion scintigraphy, in the side of the reoperation.

Immature teratoma of the uterine fundus.

An exceedingly rare case of extragonadal immature teratoma, which occurred primarily in the uterus, is described. The tumor developed into the pelvic cavity from the uterine fundus and consisted of ectodermal, mesodermal and endodermal derivatives. There were also significant amounts of immature elements; immature neuroepithelium with brisk mitotic activity, immature mesenchymal tissue, immature cartilage, immature striated muscle and immature hepatic tissue. Histologically, it was classified as a grade 3 immature teratoma. Treatment consisted of total simple hysterectomy followed by 2 courses of combination chemotherapy with vincristine, actinomycin D and cyclophosphamide (VAC). The patient was well and without evidence of recurrence at 5 years post-operatively.

Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C.

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.

Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.