Role of angiogenic growth factors in transplant coronary artery disease

Transplant coronary artery disease (TxCAD) as a manifestation of chronic rejection is a major limitation to long‐term survival of heart transplant recipients. Although the exact molecular and cellular mechanisms contributing to neointimal formation are unknown, it has been generally believed that smooth muscle cells (SMC) of donor origin migrate from the media into the subendothelial layer of the vascular wall, where SMC proliferate and synthesize extracellular matrix resulting in intimal thickening. However, recent observations indicate that hematopoietic and vascular progenitor cells derived from recipient bone marrow may contribute to the arteriosclerotic lesion formation in the coronary arteries of the transplant. On the other hand, studies on postnatal hematopoiesis indicate that angiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin‐1 (Ang1) may regulate the recruitment of these cells into distant organs. Furthermore, embryonic VEGFR‐2 ⁺ /CD34 ⁺ stem cells may serve as vascular progenitor cells and their differentiation into endothelial cells and SMC may be regulated by VEGF and platelet‐derived growth factor (PDGF), respectively. In this review, we discuss the role of angiogenic growth factors such as VEGF, Ang, and PDGF in the recruitment of hematopoietic and vascular progenitor cells in TxCAD and suggest novel therapies targeted at homing, differentiation and proliferation of these cells in the allograft.     

Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H⁺-ATPase (V-ATPase) and the vacuolar H⁺-pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn²⁺/H⁺-antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na⁺/H⁺-antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vha-a2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth.     

Ordered membrane insertion of an archaeal opsin in vivo

The prevailing model of polytopic membrane protein insertion is based largely on the in vitro analysis of polypeptide chains trapped during insertion by arresting translation. To test this model under conditions of active translation in vivo, we have used a kinetic assay to determine the order and timing with which transmembrane segments of bacterioopsin (BO) are inserted into the membrane of the archaeon Halobacterium salinarum. BO is the apoprotein of bacteriorhodopsin, a structurally well characterized protein containing seven transmembrane alpha-helices (A-G) with an N-out, C-in topology. H. salinarum strains were constructed that express mutant BO containing a C-terminal His-tag and a single cysteine in one of the four extracellular domains of the protein. Cysteine translocation during BO translation was monitored by pulse-chase radiolabeling and rapid derivatization with a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The results show that the N-terminal domain, the BC loop, and the FG loop are translocated in order from the N terminus to the C terminus. Translocation of the DE loop could not be examined because cysteine mutants in this region did not yield a gel shift. The translocation order was confirmed by applying the assay to mutant proteins containing two cysteines in separate extracellular domains. Comparison of the translocation results with in vivo measurements of BO elongation indicated that the N-terminal domain and the BC loop are translocated cotranslationally, whereas the FG loop is translocated posttranslationally. Together, these results support a sequential, cotranslational model of archaeal polytopic membrane protein insertion in vivo.     

Evaluation of chemiluminescence immunoassays for detecting thyroglobulin (Tg) and thyroid peroxidase (TPO) autoantibodies using the IMMULITE 2000 system

The chemiluminescence assays for detection of autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg) implemented on the IMMULITE 2000 system (Diagnostic Products Corporation) were evaluated. These were immunometric assays with antigen-coated beads and monoclonal murine anti-IgG antibodies conjugated with alkaline phosphatase. Precision was satisfactory with an intraassay precision of 5.3-5.5% for anti-Tg and 4.8-5.3% for anti-TPO and an interassay precision of 5.7-7.3% for anti-TPO and 5.2-7.5% for anti-Tg. The lower detection limit was determined as 5 IU/ml for anti-TPO and 2.2 IU/ml for anti-Tg. The average dilution linearities of 102% for anti-TPO and 100% for anti-Tg and the average recovery of 80-127% for anti-TPO and 93-112% for anti-Tg were acceptable. The findings of the tests were compared with the systems from Pharmacia & Upjohn, ORGenTec, Roche Diagnostics, Byk Sangtec Diagnostica and BRAHMS Diagnostica. Taking the respective cutoff value into account, concordance was 87-96% for anti-Tg and 87-97% for anti-TPO. Summarizing all results from the different methods revealed a clinical agreement of 95% for anti-TPO and 93% for anti-Tg. A good agreement was found with the IMMULITE anti-TPO and anti-Tg assays, which are closely related as regards method and biochemistry. Regression analysis gave the following results: anti-TPO IMMULITE 2000 vs anti-TPO IMMULITE: anti-TPO IMMULITE 2000 = 0.99 x IMMULITE anti-TPO - 1.43 IU/ml (r = 0.99, n = 144). anti-Tg IMMULITE 2000 vs anti-Tg IMMULITE: anti-Tg IMMULITE 2000 = 0.98 x IMMULITE anti-Tg + 1.63 IU/ml (r = 0.99, n = 86). Further age-dependent normal ranges were evaluated. A higher prevalence of elevated autoantibody titers was found for patients older than 50 years. The rate of elevated antibody titer can be reduced by using an age-dependent reference range: < or = 50 years anti-TPO < 35 IU/ml, anti-Tg < 40 IU/ml and > 50 years anti-TPO < 100 IU/ml, anti-Tg < 80 IU/ml. Further samples from clinically diagnosed Hashimoto's thyroiditis and Graves' disease were investigated. The levels of positive anti-Tg values and anti-TPO values accorded with those stated in the literature and were comparable to those measured with a reference assay. In the tested INSTAND e. V. interlaboratory samples there was high-level accordance with the expected clinical results.     

Chronic ulcerations following topical therapy with 5-fluorouracil for vaginal human papillomavirus-associated lesions.

Applied topically to the vagina, 5-fluorouracil (5-FU) cream is an effective therapy for human papillomavirus (HPV)-associated lesions of the vagina including condylomata acuminata and vaginal intraepithelial neoplasia. Although the acute side effects of 5-FU therapy are well recognized, long-term sequelae of intravaginal 5-FU use have not been described in detail in the literature. To assess the incidence and clinical course of 5-FU-related vaginal mucosal alterations, we studied 220 patients who underwent 5-FU therapy for HPV-associated lesions of the vagina. Eighteen women (8.2%) had epithelial ulcers 6 months after completion of the 5-FU treatment. The incidence of ulcers was higher in women who used 5-FU for longer than 10 weeks compared with those who used it for 10 weeks or less (9.6 versus 5.7%; P = .05). All but one of the mucosal defects were in the vaginal fornices and/or the periphery of the ectocervix. The ulcers were mostly singular and measured 0.5-7 cm in greatest diameter. Fourteen patients (77.8%) had symptoms related to the ulcers including a serosanguineous or watery discharge (55.6%), postcoital spotting or bleeding (44.4%), irregular bleeding unrelated to intercourse (16.7%), and pain (5.6%). Spontaneous healing of the ulcers was protracted. Office methods of therapy including estrogen creams and cauterizing agents failed to accelerate healing as compared with untreated patients. Excision of the ulcer and primary closure of the wound was curative in all four cases in which it was used. We conclude that topical 5-FU therapy may lead to troublesome chronic mucosal ulcers that tend to persist despite conservative treatment attempts.     

Cellular sensitization against spermatic and seminal plasma antigens in women after intrauterine insemination

Summary Using an indirect lymphokin-assay, the leucocyte-migration-inhibition-test (LMI-test), the cellular sensitization of fertile and infertile patients before and after homologous and heterologous intrauterine insemination (IUI) was investigated. In this assay several preparations of spermatozoa (“washed”-, “swim-up”- and “pellet”-spermatozoa) in different concentrations (1, 5 and 10×10⁶ sperms/ml culture medium) and seminal plasma were tested as antigen. In all investigated groups a cellular immune response against spermatic antigen was demonstrable and seemed to be dose dependent. In contrast to fertile women who reacted with an enhancement of the macrophage migration for low concentrations the same concentration of antigen induced an inhibition of macrophage migration in fertile patients. For high concentrations of spermatic antigens there was a difference in the intensity of cell-mediated immune response between fertile and infertile women. Since infertile patients demonstrated an increased level of cell-mediated immune response it is possible that infertility may be caused by this altered immunological reaction. This response changes after multiple IUI-treatment and that change might be caused by the high concentration of spermatic antigens as there was a difference in the intensity of cell-mediated immune response between fertile and infertile women. Since infertile patients demonstrated an increased level of cell-mediated immune response it is possible that infertility may be caused by this altered immunological reaction. This response changes after multiple IUI-treatment and that change might be caused by the high concentration of spermatozoa. The immunological response of infertile patients seems to be similar in those receiving husband and donor IUI.     

Whole abdominal radiotherapy following cytoreductive surgery and chemotherapy in ovarian carcinoma

Persistent or recurrent disease following surgery and chemotherapy in ovarian carcinoma remains a major therapeutic dilemma. Between January 1980 and December 1985, there were 26 patients who had previously undergone cytoreductive surgery and chemotherapy and were treated with external beam radiotherapy. Twenty-one of these patients had been treated with platinum-adriamycin-cytoxan (PAC) regimen and 5 were treated with other combinations. Surgical reevaluation was performed in 21 of the 26 patients and only 4/21 (19%) patients were free of disease. All 26 patients were irradiated with a planned dose of 2500 cGy/100 cGy/day or 2280 cGy/120 cGy/day to the whole abdomen and a final calculated dose to the pelvis of 4500 cGy. Initial evaluation showed a 3-year actuarial survival rate of 51% and a disease-free survival rate of 42%. Follow-up analysis yields survivals of 45 and 35%, respectively. Severe gastrointestinal complications were observed in 3/26 patients and all hematologic complications resolved. Variables of prognostic significance were chemotherapy tolerance, grade, and volume of residual disease. We conclude that a proportion of patients with disease following cytoreductive surgery and chemotherapy may be salvaged with abdominopelvic irradiation.     

Intrapartum fetal heart rate monitoring. VIII. Atypical variable decelerations

A total of 1,996 fetal heart rate (FHR) tracings were analyzed to assess the prognostic significance of variable decelerations. Nineteen percent (186 cases) of 988 tracings with variable decelerations in the last 30 minutes of monitored labor exhibited signs of atypia listed in order of frequency: (1) loss of initial acceleration, (2) slow return to the baseline FHR, (3) loss of secondary acceleration, (4) prolonged secondary acceleration, (5) biphasic deceleration, (6) loss of variability during deceleration, and (7) continuation of the baseline at a lower level. Variable decelerations with one or more of these features were called atypical variable decelerations and predicted a high incidence of fetal acidosis and low Apgar scores. By contrast, adverse fetal outcome was uncommon with pure variable decelerations (p much less than 0.001) irrespective of the duration and amplitude of the deceleration. Both pure and atypical variable decelerations were associated with other FHR abnormalities in over 60% of the cases. However, the particularly unfavorable combination with decreased FHR variability and tachycardia or bradycardia was seen more frequently with atypical than with pure variable decelerations (p much less than 0.001) and predicted the highest incidence of low Apgar scores. It is concluded that atypical features aid greatly in the identification of distress in fetuses with variable decelerations.