Mechanisms responsible for delayed and immediate hemolytic transfusion reactions in a patient with anti-E + Jk(b)+ Di(b) and anti-HLA alloantibodies

Immediate hemolytic transfusion reactions (IHTR) occurred in the course of delayed hemolytic transfusion reactions (DHTR). An 84-year-old man had received a blood transfusion 20 years ago. Progressive anemia developed, because of continuous bleeding from a bladder tumor. He was transfused with concentrated red blood cells (CRC) which were Rh-E antigen negative, because he had anti-E antibodies (day 0). He received CRC on day 3, and underwent resection of bladder tumor on day 6. Although crossmatch-compatible CRCs were prepared for the operation, those were not required and were kept in a refrigerator in the ward. On day 9, when a CRC kept in the ward was transfused, he suddenly had a IHTR. In order to analyze a mechanism of IHTR, the anti-Jk(b) and anti-Di(b) antibodies, anti-HLA antibodies and the concentrations of inflammatory cytokines were measured in serum samples. The anti-Jk(b) and anti-Di(b) antibodies increased prior to IHTR experienced on day 9. The concentrations of IL-6 and IL-1beta increased from day 2, while the concentration of IL-8 increased from day 7. The anti-HLA class I antibody could be detected 2 days before IHTR. Thus, the anti-Jk(b) and anti-Di(b) antibodies induced the production of inflammatory cytokines and symptoms of DHTR and IHTR. The anti-HLA class I antibody could be produced in spite of using the filer for removing leukocytes, and may take part in the induction of IHTR. Further, blood products should be transfused soon after completing a crossmatch test in patients with anti-RBC alloantibodies.     

Apoptosis-inducing protein derived from hepatocyte selectively induces apoptosis in lymphocytes

The liver is where lymphocytes undergo activation-induced cell death (AICD) at the resolution phase of an immune response, which is crucial for homeostasis of the immune system and prevention of autoimmunity. Exploring the machinery of AICD in the liver, we found that a primary culture supernatant of murine hepatocytes had an antiproliferative effect on antigen-stimulated T clone and T lymphoma cells. Biological study showed that the antiproliferation was due to induction of apoptosis in a caspase-dependent manner. The apoptosis-inducing potential was sensitive to trypsin, heat (> 70 degrees ) and acid (< pH 5) treatment but could not be neutralized by anti-tumour necrosis factor-alpha, anti-Fas ligand, or anti-transforming growth factor-beta antibodies. Biochemical study of the isolated and purified apoptosis-inducing component from the supernatant showed that it was a protein with a molecular mass of about 68,000-70,000. It induced apoptotic change in murine T and B cells, and to a lesser degree, in human lymphoid cells, but not in macrophages. Biochemical and biological characteristics distinguish this protein from others that have been reported to induce apoptosis of lymphocytes. The identification of an apoptosis-inducing protein derived from murine hepatocytes, which selectively induces apoptosis in lymphocytes, suggests one possible mechanism for immune suppression in the liver.     

Light and electron microscopic analyses of immediate and late tissue damage caused by radiofrequency ablation in porcine liver

Radiofrequency ablation (RFA) is an effective procedure for localized hepatocellular carcinoma. Contrast-enhanced CT depicts the ablated area as a hypoattenuated area without hepatic blood flow; however, light microscopy does not show obvious necrosis in the ablated area. We evaluated liver tissue changes after RFA by light microscopy and electron microscopy. The normal livers of three anesthetized pigs were coagulated using RFA after laparotomy. The liver was examined immediately, and 1 week after operation by light and electron microscopy. After RFA, the liver parenchyma surrounding the needle electrode was brown in color and surrounded by a red marginal zone separate from the normal liver parenchyma. Hematoxylin-eosin staining of the central area did not show cell necrosis, and the structures of liver sinusoids, liver cell cord and the nuclei of hepatocytes were preserved. However, electron microscopic examination of tissue immediately after RFA showed destruction of mitochondria of hepatocytes and fixation of sinusoidal cells. One week later, there was a large quantity of debris in the enlarged sinusoids, in addition to irreversible destruction of hepatocyte organelles. RFA of the porcine liver causes hepatocyte damage. This damage was not evident by light microscopy but clearly identified by electron microscopy.     

Electro-acupuncture stimulation effects on duodenal motility in anesthetized rats

The effect of electro-acupuncture stimulation (EAS) on duodenal motility was examined in anesthetized, artificially ventilated rats. EAS was applied to the abdominal area or to a hindpaw for 30 s at stimulus intensities of 0.1-10.0 mA with a stimulus frequency of 20 Hz. The duodenal motility was measured using the balloon method at a position about 1.5 cm caudal from the pylorus. Duodenal motility was inhibited by EAS at intensities of more than 5.0 mA (suprathreshold of group IV afferent excitation) when applied to the abdominal area. The duodenal inhibitory response existed after bilateral vagotomy or spinal transection, but was abolished by sectioning bilateral splanchnic nerves. Duodenal motility was facilitated by EAS at intensities of more than 2.0 mA (subthreshold of group IV, and suprathreshold for groups II+III afferent excitation) when applied to a hindpaw. The duodenal facilitatory response by EAS to a hindpaw existed after sectioning the splanchnic nerves, but disappeared after bilateral vagotomy or spinal transection. Furthermore, repetitive electrical stimulation of vagal efferent nerves enhanced duodenal motility, while repetitive electrical stimulation of the splanchnic efferent nerves inhibited the motility. It was concluded that the inhibitory response of duodenal motility elicited by EAS to the abdominal area is a spinal reflex response involving splanchnic inhibitory efferent nerves, and the enhanced response of duodenal motility by EAS to a hindpaw is a supraspinal reflex response involving vagal excitatory nerves.     

The effect of peroxisome proliferator-activated receptor-gamma ligand on urological cancer cells

Peroxisome proliferator activator-receptor (PPAR)-gamma ligand induces growth arrest of cancer cells through apoptosis. In this study, we examined the effects of PPAR-gamma inhibitors on cell proliferation in renal cell carcinoma (RCC), bladder tumor (BT), and prostatic carcinoma (PC) cell lines. We investigated the inhibitory effect of PPAR-gamma ligands, troglitazone and 15-deoxy-Delta12,14-prostaglandin J2 (15dPGJ2) on RCC, BT and PC-derived cell lines using MTT assay and Hoechst staining. PPAR-gamma ligands (troglitazone and 15dPGJ2) induced the reduction of cell viability with the half-maximal concentration of growth inhibition of RCC, BT, and PC cell lines. Furthermore, counting cells at days 1, 2 and 3, clearly showed marked inhibition of cell proliferation using troglitazone and 15dPGJ2. All PPAR-gamma inhibitors stopped the growth of all RCC, BT and PC cells. Cells treated with PPAR-gamma inhibitors showed chromatin condensation, cellular shrinkage, small membrane-bound bodies (apoptotic bodies), and cytoplasmic condensation. These cellular changes were typically redundant characteristics of apoptosis. PPAR-gamma ligands may mediate potent antiproliferative effects against RCC, BT and PC cells through differentiation. Thus, PPAR-gamma may become a new target in treatment of urological tumors.     

Exacerbation of rheumatoid arthritis following Helicobacter pylori eradication: disruption of established oral tolerance against heat shock protein?

A 62-year-old Japanese woman with RA received an eradication therapy against Helicobacter pylori in November 1999. Eight weeks later, successful eradication was confirmed by negative results for rapid urease test, pathologic findings, and a fall in anti-H. pylori IgG antibody titer. During the course, parameters for RA activity were exacerbated: C-reactive protein 1.1-4.2 mg/dL, rheumatoid arthritis precipitation antigen 2560-5120 dils., erythrocyte sedimentation rate 52-123 mm/h, and complements CH50 50 to over 60 U/mL. Lansbury index increased from 70% to 105%. Two more weeks later, the patient noticed right shoulder pain. She also complained of bilateral gonalgia two months later, and physical examination revealed increased fluid in the knee joints. Prednisolone was required to control the disease activity. The results of this case suggested that RA patients might experience a deleterious effect on the disease activity following H. pylori eradication possibly through disruption of the established oral tolerance against stress protein such as mycobacterial heat shock protein 65.     

Heat-shock induced nuclear retention and recycling inhibition of importin alpha

Heat-shock induces a strong stress response and modifies all aspects of cellular physiology, which involves dynamic changes in the nucleocytoplasmic distributions of a variety of proteins. Many distinct nucleocytoplasmic transport pathways exist in eukaryotic cells, but how a particular transport pathway is regulated under different cellular conditions remains elusive. The finding of this study indicate that conventional nuclear import, which is mediated by importin alpha/beta, is down-regulated, while the nuclear import of 70 kD heat-shock cognate protein is up-regulated in heat-shock cells. Among the factors involved in the mediation of the conventional nuclear import, significant levels of importin alpha accumulate in the nucleus in response to heat-shock. An analysis of the behaviour of importin alpha with fluorescence recovery after photobleaching and fluorescence loss in photobleaching studies show that nuclear importin alpha becomes less mobile and its nucleocytoplasmic recycling is impaired in heat-shock cells. These data coincided well with biochemical and cytological studies. Our present data show that heat-shock induces the nuclear accumulation, nuclear retention, and recycling inhibition of importin alpha, resulting in the suppression of conventional nuclear import. This suggests a new regulatory mechanism for the adaptation of cells to environmental changes, such as heat-shock.     

Depletion of mitochondrial DNA in HIV-1-infected patients and its amelioration by antiretroviral therapy

Mitochondrial DNA (mtDNA) of peripheral blood mononuclear cells (PBMCs) collected from Human immunodeficiency virus 1 (HIV-1)-infected patients and healthy controls were measured longitudinally using real-time polymerase chain reaction to evaluate the effects of antiretroviral agents on mtDNA synthesis in vivo and to assess the value of monitoring mtDNA in PBMCs to predict adverse events amongst these patients. MtDNA levels in PBMCs were significantly decreased in treatment-naive HIV-1-infected patients compared with healthy people. MtDNA levels were not only significantly correlated with CD4(+) T-cell count, but also inversely correlated with HIV-1 viral load. MtDNA levels in untreated patients and healthy controls were stable during the period of observation. On the other hand, amongst patients treated with regimens containing AZT/3TC or d4T/3TC, mtDNA increased during treatment and recovered to levels comparable to healthy controls. In contrast, mtDNA decreased immediately after the initiation of an AZT/ddC-containing regimen. We did not find a correlation between mtDNA levels and changes in clinical parameters. There was no significant difference in mtDNA levels between patients with and those without lipoatrophy. Furthermore, there was no obvious difference in mtDNA levels amongst those patients exhibiting signs and symptoms of peripheral neuropathy. In conclusion, the decrease in mtDNA levels in PBMCs amongst HIV-1-infected patients and its amelioration by antiretroviral therapy may suggest the influence of direct effects on mitochondria or mtDNA by HIV-1 infection. Further investigations are needed to elucidate the mechanisms contributing to decreased mtDNA and the value of mtDNA measurement in the care of HIV-1-infected individuals.     

Use of an Enrichment Broth Cultivation-PCR Combination Assay for Rapid Diagnosis of Swine Erysipelas

We have previously described the creation by Tn916 mutagenesis of avirulent transposition mutants from a highly virulent strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas. In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of E. rhusiopathiae. According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the E. rhusiopathiae chromosome by PCR. The specificity of the PCR was investigated by analyzing 64 strains of Erysipelothrix species and 27 strains of other genera different from Erysipelothrix. A 937-bp DNA fragment could be amplified from all E. rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested. To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify E. rhusiopathiae in the specimens from swine with arthritis. After samples were enriched in selective broth culture, detection of E. rhusiopathiae was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR was 10³ CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E. rhusiopathiae in specimens.