Identification of uromodulin deposition in the stroma of perinephric fibromyxoid nephrogenic adenoma by mass spectrometry.

Nephrogenic adenoma (NA) is an epithelial lesion that usually occurs in the mucosa of the urinary tract. Rare cases of deep infiltrative or perinephric lesions have also been reported. Recently, NA with characteristic fibromyxoid stroma (fibromyxoid NA) has been proposed as a distinct variant. Although shedding of distal renal tubular cells due to urinary tract rupture has been postulated as the cause of NA in general, the mechanism underlying extraurinary presentation of NA and fibromyxoid stromal change in fibromyxoid NA remains unknown. In this study, we performed mass spectrometry (MS) analysis in a case of perinephric fibromyxoid NA of an 82-year-old man who underwent right nephroureterectomy for distal ureteral cancer. The patient had no prior history of urinary tract injury or radiation. Periodic acid-Schiff staining-positive eosinophilic structureless deposits in the stroma of fibromyxoid NA were microdissected and subjected to liquid chromatography/MS. The analysis revealed the presence of a substantial amount of uromodulin (Tamm-Horsfall protein). The presence of urinary content in the stroma of perinephric fibromyxoid NA suggests that urinary tract rupture and engraftment of renal tubular epithelial cells directly cause the lesion.

Disorganization of Oligodendrocyte Development in the Layer II/III of the Sensorimotor Cortex Causes Motor Coordination Dysfunction in a Model of White Matter Injury in Neonatal Rats.

We previously established neonatal white matter injury (WMI) model rat that is made by right common carotid artery dissection at postnatal day 3, followed by 6% hypoxia for 60 min. This model has fewer oligodendrocyte progenitor cells and reduced myelin basic protein (MBP) positive areas in the sensorimotor cortex, but shows no apparent neuronal loss. However, how motor deficits are induced in this model is unclear. To elucidate the relationship between myelination disturbance and concomitant motor deficits, we first performed motor function tests (gait analysis, grip test, horizontal ladder test) and then analyzed myelination patterns in the sensorimotor cortex using transmission electron microscopy (TEM) and Contactin associated protein 1 (Caspr) staining in the neonatal WMI rats in adulthood. Behavioral tests revealed imbalanced motor coordination in this model. Motor deficit scores were higher in the neonatal WMI model, while hindlimb ladder stepping scores and forelimb grasping force were comparable to controls. Prolonged forelimb swing times and decreased hindlimb paw angles on the injured side were revealed by gait analysis. TEM revealed no change in myelinated axon number and the area g-ratio in the layer II/III of the cortex. Electromyographical durations and latencies in the gluteus maximus in response to electrical stimulation of the brain area were unchanged in the model. Caspr staining revealed fewer positive dots in layers II/III of the WMI cortex, indicating fewer and/or longer myelin sheath. These data suggest that disorganization of oligodendrocyte development in layers II/III of the sensorimotor cortex relates to imbalanced motor coordination in the neonatal WMI model rat.

Studies of assisted reproduction techniques (ART) for HIV-1-discordant couples using washed sperm and the nested PCR method: a comparison of the pregnancy rates in HIV-1-discordant couples and control couples.

In this study, the efficacy and safety of assisted reproduction techniques with the sperm-washing method and nested PCR assay were evaluated in HIV-1-discordant couples, as many HIV-1-positive people of reproductive age are getting married and wish to have children safely. Twenty-seven HIV-1-discordant couples (husband, positive; wife, negative) were enrolled in this study. The spermatozoa were separated from semen samples by density gradient centrifugation and the swim-up method. HIV-1 RNA and proviral DNA were checked using nested PCR with a detection limit of one copy before fertilization and before embryo transfer. Clinical outcomes were compared with those of matched control couples. Thirty-eight cycles of in vitro fertilization or intracytoplasmic sperm injection were performed in HIV-1-discordant couples, where the pregnancy rates per embryo transfer and per couple were 60.6 and 63.0%, respectively. These rates were significantly higher than those in control couples (P<0.05). Furthermore, all of the females and babies remained HIV-1 negative throughout the study period. Our data strongly suggest that this technique will allow HIV-1-discordant couples to conceive more safely and effectively.

Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening.

CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-gamma) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-gamma by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-gamma and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.

Glutamine Deprivation Enhances Acetyl-CoA Carboxylase Inhibitor-induced Death of Human Pancreatic Cancer Cells.

BACKGROUND/AIM: Acetyl-CoA carboxylase (ACC) is a rate-limiting enzyme in fatty acid synthesis. In this study, we investigated the effect of ACC inhibition on survival of pancreatic cancer cells. MATERIAL AND METHODS: AsPC-1, BxPC-3 and PANC-1 were used as human pancreatic cancer cell lines. 5-(etradecyloxy)-2-furoic acid (TOFA) and bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES) were used as inhibitors of ACC and glutaminase (GLS) respectively. Apoptotic and live cells were distinguished by annexin-V staining. The activity of caspase-3 was evaluated by measuring the fluorescence intensity of the degradation product of the substrate, N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin. RESULTS: TOFA increased the number of annexin V-positive cells and enhanced caspase-3 activity in AsPC-1 and BxPC-3, but not in PANC-1 cells. The number of PANC-1 cells increased after 48 h in Earle's balanced salt solution. Interestingly, proliferation of PANC-1 cells was drastically suppressed by glutamine deprivation, but not by inhibition of glycolysis. BPTES also induced cell death to the same extent as glutamine deprivation. In addition, TOFA induced cell death of PANC-1 cells, both in the presence of BPTES and with glutamine deprivation, suggesting that inhibition of glutaminolysis causes cell death and enhances the effect of TOFA in PANC-1 cells. CONCLUSION: These findings suggest that glutaminolysis is important for the survival of pancreatic cancer cells showing tolerance to nutrient starvation such as PANC-1 cells, and use of a combination of inhibitors of ACC and GLS may be a new strategy for treatment of pancreatic cancer.

A headache-free reversible cerebral vasoconstriction syndrome (RCVS) with symptomatic brain stem ischemia at late pregnancy as a rare manifestation of RCVS resolved with termination of pregnancy by semi-urgent cesarean section.

A 32-year-old pregnant woman in her 39th week of pregnancy presented at the emergency room complaining of sudden-onset dizziness with gaze disturbance and was admitted to our hospital. Her past medical history included hypertension, diabetes mellitus and infarction in the right medulla oblongata 18 months prior to this event. Magnetic resonance (MR) angiography showed multiple irregular stenosis of the intracranial arterial system. Although MR images revealed no fresh ischemic or hemorrhagic lesions, she was diagnosed with reversible cerebral vasoconstriction syndrome (RVCS) associated with pregnancy. Cesarean section immediately resolved the headache-free ischemic RCVS. The postpartum course of the patient was uneventful as well as that of her baby. Follow-up MR angiography showed improvement of intracranial vasoconstriction and follow-up MR imaging showed improvement of a left medial pontine ischemic lesion on diffusion-weighted image. This report describes a rare manifestation of pregnancy-related RCVS.

Complete removal of HIV-1 RNA and proviral DNA from semen by the swim-up method: assisted reproduction technique using spermatozoa free from HIV-1.

BACKGROUND: Use of antiretroviral drugs has reduced the mortality rate for HIV infection and many HIV-discordant couples wish to have children. It is possible for an HIV-infected man to father children without risk of HIV transmission if HIV-free spermatozoa can be obtained from his semen. METHODS: An improved swim-up method was used to collect HIV-free spermatozoa from the semen of HIV-positive males. Diluted semen was layered over a Percoll solution with a continuous density gradient of 30-98%, and then centrifuged. The bottom layer was collected by cutting the end from the tube and the sperm suspension was collected using the swim-up method. Spermatozoa were tested by nested polymerase chain reaction (PCR) for HIV-1 RNA and DNA, with a detection limit of one copy. Spermatozoa were used for assisted reproduction in 43 couples. RESULTS: HIV-1 RNA and proviral DNA were not detected by nested-PCR assay in all 73 of the collected spermatozoa samples from 52 patients. The HIV-1-negative sperm was used for in vitro fertilization in 12 couples and for intracytoplasmic sperm injection in 31 couples. No detection of HIV-1 RNA or proviral DNA in the culture medium of the fertilized eggs was confirmed again before embryo transfer. Of the 43 female partners, 20 conceived and 27 babies were born. HIV antibodies, HIV RNA and proviral DNA were negative in all of the females and babies. CONCLUSIONS: HIV-negative spermatozoa could be obtained from semen of HIV-positive men. The method involves no risk of HIV transmission to female partners and their children.

Prognostic factors for ovulatory response with clomiphene citrate in polycystic ovary syndrome.

OBJECTIVE: To evaluate prospectively the prognostic factors for ovulatory responses following clomiphene citrate (CC) administration in polycystic ovary syndrome (PCOS). STUDY DESIGN: Fifty-nine infertile patients with a diagnosis of PCOS were recruited. Ovulation was induced using 100 mg/day CC administered daily from days 5 to 9 of the cycle. Endocrine and metabolic parameters between responder and non-responder groups were analyzed. RESULTS: For a 75-g fasting glucose load (75-g OGTT), blood glucose levels at 60 and 120 min, the area under the curve (AUC) and blood insulin levels at 120 min in the non-responder group (n=25) were significantly higher than those in the responder group (n=34), although the measurements of fasting blood glucose and insulin were not significantly different between the two groups. In the receiver operating characteristic curves, the most appropriate cutoff point was 120 mg/dl for the blood glucose level at 120 min and 9000 for the blood glucose x insulin level at 120 min. There were no significant differences in the clinical characteristics or in the endocrine and metabolic parameters between conceived (n=9) and non-conceived groups (n=21). CONCLUSION: The levels of blood glucose and blood glucose x insulin at 120 min after 75-g OGTT could be good biochemical markers of CC resistance in PCOS. No predictors of conception following CC therapy were identified by this study.

Dysfunction in Motor Coordination in Neonatal White Matter Injury Model Without Apparent Neuron Loss.

We made a white matter injury (WMI) model with mild hindlimb dysfunction by right common carotid artery occlusion followed by 6% oxygen for 60 min at postnatal day 3 (P3), in which actively proliferating oligodendrocyte (OL) progenitors are mainly damaged. To know whether this model is appropriate for cell therapy using OL progenitors, the pathological response to mild hypoxia-ischemia (H-I) in neurons and OL lineage cells and myelination failure were investigated along with gene expression analysis. In WMI model rats, coordinated motor function, as assessed by the accelerating rotarod test, was impaired. The dysfunction was accompanied by myelination failure in layers I-IV of the sensorimotor cortex. Although several oligo2-positive OLs stained positive for active caspase 3 in the cortex and white matter at 24 h after H-I, few NeuN-positive neurons were apoptotic. Argyrophil-III staining for damaged neurons revealed no increase in the number of degenerating cells in the model. Moreover, the total number of NeuN-positive neurons in the cortex was comparable to that of controls 7 days later. Retrograde labeling of the corticospinal tract with Fluoro-Gold revealed no significant loss of layer V neurons. In addition, no decrease in the numbers of cortical projecting neurons and layers V-VI neurons in both motor and sensory areas was observed. Interestingly, the numbers of inhibitory GABAergic cells immunoreactive for parvalbumin, calretinin, or somatostatin were preserved in the P26 cortex. Gene expression analysis at P5 revealed 98 upregulated and 65 downregulated genes that may relate to cell survival, myelin loss, and differentiation of OLs. These data suggest that impaired motor coordination was not induced by neuron loss but, rather, myelination failure in layers I-IV. As OL lineage cells are mainly damaged, this WMI model might be useful for cell-based therapy by replacing OL progenitors.

Expression mapping using a retroviral vector for CD8+ T cell epitopes: definition of a Mycobacterium tuberculosis peptide presented by H2-Dd.

Identification of CD8+ T cell epitopes is important because detection of specific CD8+ T cells after infection or immunization requires prior knowledge of epitope specificity. Furthermore, identification of CD8+ T cell epitopes permits the development of specific preventive and therapeutic approaches to both infections and tumors. Thus far, CD8+ T cell epitopes have been identified either using an overlapping peptide library covering an entire protein, or using algorithms designed to identify likely peptides that bind to major histocompatibility complex (MHC) class I molecules. The synthesis of overlapping peptides can be prohibitively expensive, and the algorithm programs used to predict CD8+ T cell epitopes are not always accurate. Here we describe a retroviral expression system that specifically allows longer polypeptides and shorter peptides to be expressed in the cytoplasm, and thereby to be processed onto class I MHC molecules. T cells from mice that were immunized with a DNA vaccine encoding MPT-51 were probed against MHC-compatible cell lines retrovirally transduced with overlapping gene fragments encoding 120-140 amino acids of the MPT-51 molecule. After further testing of shorter peptide sequences, we identified a CD8+ T cell epitope using cell lines expressing a relatively small number of algorithm-predicted candidate epitopes. We found that one of the requirements for cell surface display of the 20-mer peptide was the need for cotranslational ubiquitination. The restriction molecule was identified as Dd following transduction with MHC class I genes followed by transduction with the oligonucleotide encoding the epitope. The retroviral expression system described here is cost-effective, particularly if the target molecule is large, and could be adapted to identifying T cell epitopes recognized in infectious disease and against tumor cell antigens.

A five-year review of patients admitted with the diagnosis of bacterial endophthalmitis.

We conducted a retrospective, hospital-based study of patients who were diagnosed with bacterial endophthalmitis on admission to the Department of Ophthalmology, Hiroshima University Hospital, between January 1999 and December 2003. Thirty eyes of 30 patients were identified. Of these patients, 19 eyes had postoperative endophthalmitis, 8 eyes had penetrating trauma and 3 eyes were infected from an endogenous source. All of the patients underwent immediate three-port pars plana vitrectomy. Vitreous specimens of diabetic patients demonstrated a significantly higher incidence of positive bacterial culture. Diabetic vitreous appeared to be a good medium for culture. Visual acuity of hand motion or less at the latest follow-up visit was associated with the presence of diabetes. Prompt treatment with vitrectomy and intra-vitreal antibiotics is crucial for patients with bacterial endophthalmitis, especially if they are diabetic.

Cytotoxic T-lymphocyte-, and helper T-lymphocyte-oriented DNA vaccination.

DNA vaccines have advantages over other types of vaccines in that they can induce strong cellular immune responses, namely cytotoxic T lymphocytes (CTL) and helper T lymphocytes (Th). DNA vaccines are therefore considered a promising alternative to attenuated live vaccines in the field of infectious diseases. So far, various DNA vaccines have been generated and tried to induce a particular cellular immune response by virtue of recombinant DNA technology. DNA vaccines have been designed for efficient transcription and translation of target genes by a variety of strategies. Also, various DNA vaccine strategies for induction of specific CTL and Th have been reported by taking into consideration antigen presentation pathways and the strategies have been shown to be effective to elicit particular T-cell responses. In this paper, we have reviewed these strategies, including our study on epitope-specific T-cell induction by DNA vaccination against Listeria monocytogenes infection. From this review, it has been surmised that, to induce strong immune responses by DNA vaccines, the immunization route and the immunization regimen, such as heterologous "prime-boost" regimen, should also be considered.

Effects of antioxidant treatment in oligozoospermic and asthenozoospermic men.

OBJECTIVE: To evaluate the effect of sairei-to, an herbal medicine, as an antioxidant in oligozoospermia and asthenozoospermia (nonnormozoospermia). STUDY DESIGN: Forty-seven nonnormozoospermic and 16 normozoospermic men were the subjects of this prospective clinical study. After sairei-to (9.0 g/d) was administered daily to the 2 groups for 3 months, sperm parameters, serum hormones and superoxide dismutase (SOD) activity in the serum and the seminal plasma was analyzed. The testicular artery was also assessed. RESULTS: After therapy, serum hormones and SOD activity did not change significantly in either group. Although men with normozoospermia did not undergo a significant change in sperm conditions or testicular artery flow, total sperm concentration (17.1 +/- 20.0 versus 28.7 +/- 35.5 x 10(6)/mL, P = .02) and sperm motility (30.1% +/- 21.6 versus 45.8% +/- 24.4, P < .0001) were significantly increased, and the pulsatility index of the testicular artery (2.03 +/- 0.84 versus 1.64 +/- 0.48, P = .04) was significantly decreased in nonnormozoospermia. CONCLUSION: Treatment with the herbal antioxidant sairei-to improves sperm condition and testicular artery flow in nonnormozoospermia.

Endometrial abnormalities in infertile women.

OBJECTIVE: To analyze retrospectively the frequency, long-term prognosis and pregnancy rate in infertile women diagnosed with endometrial hyperplasia or carcinoma by endometrial biopsy. STUDY DESIGN: From 1989 to 2000, endometrial biopsies were performed on 2,573 patients to investigate the cause of infertility. The main outcome measures were frequency, long-term prognosis and pregnancy rate for patients with each type of endometrial abnormality. RESULTS: Twenty-four patients (0.93%) were diagnosed with an endometrial abnormality. Of them, 10 were diagnosed with simple hyperplasia, 7 with complex hyperplasia, 3 with complex hyperplasia with atypia and 4 with endometrial carcinoma. All 4 patients (0.16%) with endometrial carcinoma were infertile as a result of complications arising from polycystic ovary syndrome. Two of them underwent hysterectomies. High-dose medroxyprogesterone acetate therapy combined with assisted reproductive technology resulted in pregnancy in 1 of the 2 patients with endometrial carcinoma. CONCLUSION: Of infertile women, 0.93% have endometrial abnormalities, and those with polycystic ovary syndrome have a high risk of endometrial carcinoma. Assisted reproductive technology combined with high-dose medroxyprogesterone acetate may be effective means of overcoming infertility, allowing women with endometrial carcinoma to readily achieve pregnancy.

Eyelid lengthening combined with penetrating keratoplasty for exposure keratopathy in Graves' ophthalmopathy--a case report.

Upper eyelid retraction is a well-known component of Graves' disease. With greater degrees of retraction, corneal exposure is usually increased. We report here on a patient with corneal perforation following exposure keratopathy due to upper eyelid retraction. The patient was treated with penetrating keratoplasty and an upper eyelid lengthening procedure using Goretex dura substitute as an interpositional graft material. The exposure keratopathy resolved postoperatively and this condition has been maintained for 45 months since the operation, with a good cosmetic outcome and symmetry of the palpebral fissures.

Effects of low-dose metformin in Japanese women with clomiphene-resistant polycystic ovary syndrome.

Background and Aims: The aim of the present prospective observational study was to evaluate the effects of low-dose, short-term metformin, in combination with clomiphene (CC), in CC-resistant infertile Japanese women with polycystic ovary syndrome (PCOS). Methods: Metformin therapy was administered orally (one 250 mg tablet, twice daily) to 15 CC-resistant infertile patients with PCOS, beginning on the third day of progestin-induced withdrawal bleeding, and was continued for 14 days in the first cycle. In the event of anovulation, 100 mg/day of CC was given during subsequent cycles on days 5-9, in addition to the aforementioned dose of metformin. Hormonal and metabolic parameters were measured on the second or third days of the first cycle and also the fourth cycle, following an overnight fast. Results: None of the 15 women successfully ovulated during the first cycle with metformin treatment alone. After two subsequent cycles with the combination of CC and metformin, ovulation was confirmed in 17 of 29 cycles (61%) and in 13 of 15 patients (87%). Two women became pregnant within 2 months of therapy (13%). There were no cases of ovarian hyperstimulation syndrome. Following three cycles of metformin therapy, a slight reduction in serum levels of luteinizing hormone (LH), free testosterone, androstenedione, dehydroepiandrosterone sulfate, hemoglobin A1c and total cholesterol was seen, while serum LH/follicle-stimulating hormone ratio and serum level of low-density lipoprotein cholesterol were significantly decreased. Although there were no significant differences between the responder (n = 11) and non-responder (n = 2) groups at baseline, the levels of plasma fasting insulin was significantly higher and fasting glucose/insulin ratio was significantly lower in the non-responder group compared with the responder group after three cycles. Conclusion: Low-dose, short-term metformin, combined with CC, can improve ovulation rates in CC-resistant infertile Japanese women with PCOS. (Reprod Med Biol 2004; 3: 19-26).

The loss of parts of chromosome 7 followed by the insertion of URA cassette into RB2 on MRS in Candida albicans strain CAI-4.

Clinical isolates of the medically important fungus Candida albicans show electrophoretic karyotype variations. Chromosome translocation is considered to be one of the possible mechanisms of karyotype variation and has been shown to occur very frequently at or near the unique repeated DNA sequences which comprise the Major Repeat Sequence (MRS) on the genome. The MRS consists of the repeated sequences RB2, RPS, and HOK. We previously showed the insertion at the RB2 region might initiate chromosome translocation in strain STN22u2 of C. albicans. To ask whether the insertion of a URA cassette into the RB2 but not into RPS and HOK causes chromosome translocation in C. albicans strains, we transformed three URA cassettes into strain CAI-4, which is commonly used as a host strain for gene knockout experiments. We found chromosome rearrangements followed the insertion of URA cassettes into RB2 in strain CAI-4. Three transformants had an extra chromosome showing the loss of the 7A and 7C region from one chromosome 7 homologue. The recombination occurred at or after the insertion of URA cassette into RB2. Insertion there seems to cause chromosome rearrangement and thus RB2 is considered one of the important elements for initiation of chromosome rearrangement.

Adenocarcinoma arising from respiratory ciliated epithelium in benign cystic teratoma of the ovary: a case report with analyzes of the CT, MRI, and pathological findings.

The malignant transformation of mature cystic teratoma is rare, thus occurring in only 1-2% of all cases. The most common malignancy arising in mature cystic teratoma is squamous cell carcinoma. Adenocarcinoma occurs with less frequency. We herein present a patient with an ovarian mature cystic teratoma who demonstrated a malignant transformation to well-differentiated adenocarcinoma. Malignant transformation was diagnosed preoperatively by contrast enhanced computed tomography (CT) and magnetic resonance imaging (MRI). Microscopically and immunohistochemically, the adenocarcinoma was considered to have arisen from the ciliated respiratory epithelium. After a 28-month of follow-up period, she remains free of the disease. This is the third reported case of adenocarcinoma arising in the respiratory epithelium of an ovarian mature cystic teratoma. Contrast enhanced CT and MRI are useful for making a preoperative diagnosis and an immunohistochemical study is helpful for defining its origin.