Diagnostic and therapeutic potential of tetanus toxin-derivatives in neurological diseases.

We assessed the ex vivo reactivity of peptidic constructs of Tet1 (analog of tetanus toxin non-virulent C fragment) with sequence homology to the cysteine-active site of thioredoxin (Tet1THO) or tetralysine (Tet1PLYS) with oxidative species or axonopathic sodium cyanate (NaOCN), respectively. We then assessed their neuronal uptake in vivo in laboratory animals. The reactivity of Tet1PLYS with NaOCN (1:2.5 to 1:37.5 molar ratios) or Tet1THO with hydrogen peroxide (1:0.4 to 1:6.2 molar ratios) was assessed by mass spectrometry. Green fluorescence protein (GFP)-tagged Tet1-derivatives (3 mg/ml in artificial cerebrospinal fluid) were administered daily to rats by intramuscular injection in latissimus dorsi at lumborum at the dose of 1 µl/g of body weight, for 3 days. Motor neuron uptake was assessed after double immunolabeling for GFP and choline acetyltransferase. Mass spectrometry analysis successfully demonstrated the ex vivo reactivity of Tet1-derivatives in a concentration-dependent manner. Confocal microscopy revealed the localization of Tet1-derivatives in axons and motor neuron cell bodies. Intramuscular delivery of Tet1-derivatives appears to be a practical approach to circumvent the blood nerve barrier and selectively deliver small molecules to the nervous system, for diagnostic and/or treatment purposes.

Proximal giant neurofilamentous axonopathy in mice genetically engineered to resist calpain and caspase cleavage of α-II spectrin.

We use 1,2-diacetylbenzene (1,2-DAB) to probe molecular mechanisms of proximal giant neurofilamentous axonopathy (PGNA), a pathological hallmark of amyotrophic lateral sclerosis. The spinal cord proteome of rodents displaying 1,2-DAB PGNA suggests a reduction in the abundance of α-II spectrin (Spna2), a key protein in the maintenance of axonal integrity. Protein immunoblotting indicates that this reduction is due to Spna2 degradation. We investigated the importance of such degradation in 1,2-DAB PGNA. Spna2 mutant mice lacking a calpain- and/or caspase-sensitive domain (CSD), thus hypothetically resistant to 1,2-DAB, and wild-type littermates, were treated with 1,2-DAB, 35 mg/kg/day, or saline control, for 3 weeks. 1,2-DAB induced motor weakness and PGNA, irrespective of the genotype. Spna2-calpain breakdown products were not detected in mutant mice, which displayed a normal structure of the nervous system under saline treatment. Intriguingly, treatment with 1,2-DAB reduced the abundance of the caspase-specific 120-kDa Spna2 breakdown products. Our findings indicate that degradation of Spna2 by calpain- and/or caspase is not central to the pathogenesis of 1,2-DAB axonopathy. In addition, the Spna2-CSD seems to be not required for the maintenance of the cytoskeleton integrity. Our conceptual framework offers opportunities to study the role of calpain-caspase cross talk, including that of the protease degradomics, in models of axonal degeneration.

Use of ambulatory blood pressure monitoring to compare antihypertensive efficacy and safety of two angiotensin II receptor antagonists, losartan and valsartan. Losartan Trial Investigators.

The efficacy and safety of losartan and valsartan were evaluated in a multicenter, double-blind, randomized trial in patients with mild to moderate essential hypertension. Blood pressure responses to once-daily treatment with either losartan 50 mg (n = 93) or valsartan 80 mg (n = 94) for 6 weeks were assessed through measurements taken in the clinic and by 24-hour ambulatory blood pressure monitoring (ABPM). Both drugs significantly reduced clinic sitting systolic (SiSBP) and diastolic blood pressure (SiDBP) at 2, 4, and 6 weeks. Maximum reductions from baseline in SiSBP and SiDBP on 24-hour ABPM were also significant with the two treatments. The reduction in blood pressure was more consistent across patients in the losartan group, as indicated by a numerically smaller variability in change from baseline on all ABPM measures, which achieved significance at peak (P = .017) and during the day (P = .002). In addition, the numerically larger smoothness index with losartan suggested a more homogeneous antihypertensive effect throughout the 24-hour dosing interval. The antihypertensive response rate was 54% with losartan and 46% with valsartan. Three days after discontinuation of therapy, SiDBP remained below baseline in 73% of losartan and 63% of valsartan patients. Both agents were generally well tolerated. Losartan, but not valsartan, significantly decreased serum uric acid an average 0.4 mg/dL at week 6. In conclusion, once-daily losartan 50 mg and valsartan 80 mg had similar antihypertensive effects in patients with mild to moderate essential hypertension. Losartan produced a more consistent blood pressure-lowering response and significantly lowered uric acid, suggesting potentially meaningful differences between these two A II receptor antagonists.

Hodgkin's disease in Costa Rica: a report of 40 cases analyzed for Epstein-Barr virus.

We studied 40 cases of Hodgkin's disease (HD) from Costa Rica for evidence of Epstein-Barr virus (EBV) in the Reed-Sternberg and Hodgkin (RS-H) cells. We also compared the epidemiologic features of these patients with previous reports of HD in industrialized and developing nations. Because Costa Ricans enjoy a relatively higher standard of living than the residents of other developing Central American nations yet live in the same general geographic region and are genetically similar, we believed that this comparison might shed additional light on the hypothesis that the prevalence of EBV in HD and the epidemiologic factors of HD are influenced by socioeconomic factors. In 16 (40%) of 40 cases, immunohistochemical studies demonstrated that the RS-H cells were positive for EBV latent membrane protein (LMP), including 1 case of lymphocytic depletion analyzed, 12 (86%) of 14 cases of mixed cellularity, and 3 (15%) of 20 cases of nodular sclerosis. All five cases of lymphocytic predominance were negative. In the 16 EBV LMP-positive cases, polymerase chain reaction studies revealed that the virus was type A in 12 cases and type B in 4 cases. Nodular sclerosis was the most common type of HD, accounting for 20 cases (50%), followed by mixed cellularity, with 14 cases (35%). The relatively low prevalence of EBV in the RS-H cells of HD and the high incidence of nodular sclerosis in Costa Rica are similar to industrialized nations and are unlike HD in neighboring Central American countries. These findings further support the hypothesis that the prevalence of EBV in HD and the epidemiologic features of HD are most closely linked with socioeconomic conditions, and geographic location or ethnic heritage are of relatively less importance.

Ontogeny of temperature-regulated heat shock protein 70 synthesis in preimplantation bovine embryos.

Development of the preimplantation embryo is very susceptible to disruption by heat shock. As embryos proceed through development, they acquire resistance to heat shock, perhaps because they become transcriptionally active and can respond to environmental changes by undergoing transcriptionally-regulated cellular adaptation. Objectives were to determine the ontogeny of heat shock protein 70 (HSP70) synthesis in preimplantation bovine embryos and to ascertain whether heat-induced increases in HSP70 in embryos are caused by environmental alterations in gene expression. Exposure of bovine embryos to heat shock induced synthesis of a 68 kDa form of HSP70 called HSP68 as early as the two-cell stage of development. Induction of HSP68 was alpha-amanitin independent at the two-cell stage but was blocked by alpha-amanitin as early as the early four-cell stage. Therefore, heat-induced synthesis of HSP68 is regulated at the level of transcription at a time before the major round of embryonic genome activation is considered to occur. Two other constitutive HSP70 molecules were identified called heat shock cognates (HSC) 71 and 70; both proteins were synthesized during all stages of development from the two-cell to hatched blastocyst stages. However, heat-induced synthesis of HSC71 and HSC70 was not evident until the expanded blastocyst stage. In conclusion, environmental signals can activate gene expression before the major round of embryonic genome activation occurs in bovine embryos. Moreover, differences in thermal sensitivity of early embryos to heat shock is not caused by an inability to synthesize HSP70, suggesting that other mechanisms are involved in developmental acquisition of thermotolerance.

Comparison of the usefulness of Doppler pressure half-time in mitral stenosis in patients < 65 and > or = 65 years of age.

Doppler pressure half-time is a reliable method for estimating mitral valve area when net left atrial and ventricular compliance remain stable. The accuracy of Doppler pressure half-time in estimating mitral valve area in older patients is unknown. We studied 80 patients (65 women and 15 men, aged 56 +/- 14 years) with cardiac catheterization and echocardiography. Mitral valve area was calculated using the Gorlin formula and by the Doppler pressure half-time method. Patients were stratified into those aged < 65 years (n = 57), and those aged > or = 65 years (n = 23). The discordance between pressure half-time and Gorlin-derived mitral valve area was assessed and related to multiple clinical, echocardiographic, and hemodynamic variables. The difference between pressure half-time and Gorlin-derived mitral valve area was greater in the older than in the younger patient (0.34 +/- 0.30 vs 0.15 +/- 0.27 cm2, p = 0.009) but the older group had smaller mitral valve areas by the Gorlin method (0.72 +/- 0.18 vs 0.89 +/- 0.32 cm2, p = 0.02) and lower cardiac output. The difference between pressure half-time and Gorlin remained greater in the group of older patients (0.32 +/- 0.30 vs 0.19 +/- 0.22 cm2, p = 0.04), even when the analysis was restricted to patients with similar mitral valve area (< 1 cm2 by the Gorlin method). Using multivariate analysis, age > or = 65 years remained the only significant predictor of the discrepancy between pressure half-time and Gorlin mitral valve area. Thus, when compared with Gorlin-derived mitral valve area, pressure half-time overestimated valve area in older patients, and this technique for estimating mitral valve area should be used with caution in patients > or = 65 years of age.

Subcutaneous tissue involvement by T-cell lymphoma. A report of 2 cases.

BACKGROUND: T-cell lymphomas may involve the subcutaneous tissue as a manifestation of generalized disease. However, T-cell lymphomas rarely present with extensive involvement of the subcutaneous fat without other sites of disease. OBSERVATIONS: We describe 2 women who presented with fever and subcutaneous nodules or masses. In case 1, the nodules were generalized and did not respond to chemotherapy. The patient died 2 months after diagnosis. In case 2, the mass was large but localized and responded to chemotherapy. The tumor subsequently recurred in a cervical lymph node 9 months later, and the patient was being treated with chemotherapy 15 months after initial diagnosis. Histologically, biopsy specimens from both patients revealed malignant lymphoma involving the subcutaneous tissue. The dermis and epidermis were not involved. At low power the lesions resembled panniculitis, but high-power examination revealed cytologic atypia of the malignant lymphoid cells. Immunohistochemical studies revealed T-cell lineage. In case 2, the neoplastic cells also expressed the CD30 antigen, were positive for Epstein-Barr virus RNA, and carried the t(2;5) (p23;q35) chromosomal translocation. We interpreted case 1 as an example of subcutaneous panniculitic T-cell lymphoma. We believe that case 2 is best classified as anaplastic large cell lymphoma of T-cell lineage. CONCLUSIONS: A variety of T-cell lymphomas rarely present with only subcutaneous tissue involvement. Knowledge of this phenomenon and recognition of the cytologic atypia of the lymphoid cells will help to prevent misdiagnosis.

Effect of heat shock on function of frozen/thawed bull spermatozoa.

Deposition of spermatozoa in the reproductive tract of hyperthermic cows could conceivably result in sperm damage. Accordingly, a series of experiments tested the effects of heat shock on functional characteristics and free radical production of bull spermatozoa. Viability was reduced slightly by short-term (1 to 3 h) culture at 42 and 43 degrees C as compared with culture at 39 degrees C. There was no effect of culture at 42 degrees C on the ability of spermatozoa to undergo swim-up or of 42 degrees C on the percentage of motile spermatozoa. However, exposure to 41 degrees C for 3 h reduced percentage of motile sperm, 41 and 42 degrees C reduced sperm velocity and 43 degrees C decreased the proportion of spermatozoa undergoing swim-up. In other experiments, there was no effect of heat shock (41 or 42 degrees C for 1 to 3 h) on DNA integrity, presence of intact acrosomes, or fertilizing ability of the spermatozoa. Superoxide production by spermatozoa was higher at 42 degrees C than at 39 or 41 degrees C, but there was no detectable hydrogen peroxide production at any temperature. The antioxidant, glutathione, tended to improve the ability of spermatozoa to undergo swim-up at 39 degrees C but not at 43 degrees C. Taken together, these results suggest that heat shock of a magnitude similar to that seen in vivo (41 to 42 degrees C) has little effect on sperm functions that affect fertilizing capability.

Developmental changes in sensitivity of bovine embryos to heat shock and use of antioxidants as thermoprotectants.

Experiments were conducted with in vitro-produced bovine embryos to determine whether 1) increased culture temperatures (i.e., heat shock) adversely affected embryonic development, 2) embryos become more resistant to heat shock as they advance in development, and 3) selective antioxidant molecules alleviate heat shock effects on embryonic development. Development of 2-cell embryos to > or = 16-cell stage on d 5 after in vitro fertilization was not affected by a heat shock of 40 degrees C for 3 h, but 41 or 42 degrees C for 3 h decreased (P = .004) development. In a separate experiment, development of 2-cell embryos was decreased (P = .01) by exposure to 41 degrees C for 3 h but not for 1 h. In contrast, development of morulae to blastocysts was not affected by heat shock of 41 degrees C for 1 or 3 h. Medium supplementation with 50 nM glutathione or 50 mM taurine before heat shock did not reduce the effects of heat shock (41 degrees C for 3 h) on 2-cell embryos. Likewise, addition of glutathione ester, a more membrane-permeable analog of glutathione, did not protect 2-cell embryos from heat shock. In conclusion, early bovine embryos are susceptible to disruption in development caused by heat shock. As embryos progress in development, they acquire resistance to heat shock. Glutathione, taurine, and glutathione ester were not effective in alleviating the effects of heat shock on development of 2-cell embryos. Consequently, molecules have yet to be identified that can protect early-stage bovine embryos from the adverse effects of heat shock.

Effects of interferon-tau and interferon-alpha on proliferation of bovine endometrial cells.

The ruminant conceptus secretes a unique interferon, interferon-tau, that regulates endometrial prostaglandin secretion during early pregnancy. Because one of the pleiotropic effects of interferons is to inhibit cellular proliferation, a series of experiments was conducted to determine whether or not the bovine endometrium is sensitive to the antiproliferative effect of interferon-tau and the related interferon, interferon-alpha. Endometrial epithelial and stromal cells were prepared from the endometrium of cows from Days 11-17 after estrus and incubated with recombinant bovine interferon-tau (rbIFN tau; 1-1000 ng/ml), recombinant bovine interferon-alpha 1 (rbIFN alpha; 1-1000 ng/ml), recombinant human interferon-alpha 2b (rhIFN alpha; 100 ng/ml), or ovine interferon-tau (oIFN tau; 100 ng/ml). Proliferation was determined by monitoring uptake of [3H]thymidine into DNA. Generally, interferons did not inhibit proliferation of endometrial epithelial cells. Exceptions were for 1000 ng/ml rbIFN tau, which inhibited proliferation by 23%; 100 ng/ml rbIFN alpha, which inhibited proliferation by 28% in one of two experiments only; and 100 ng/ml oIFN tau, which inhibited proliferation by 17%. Proliferation of endometrial stromal cells was not inhibited by any concentration of any interferon in two separate experiments. Therefore, unlike other bovine cells tested previously (lymphocytes and oviductal cells), bovine endometrial cells were not consistently inhibited by IFN tau or IFN alpha. Such reduced responsiveness of endometrial cells to the antiproliferative effects of type I interferons could allow for growth of the endometrium during the period of pregnancy when the conceptus produces IFN tau.

Regulation of bovine and ovine lymphocyte proliferation by progesterone: modulation by steroid receptor antagonists and physiological status.

Lectin-stimulated sheep and cow lymphocytes were used to test whether inhibitors of classical steroid receptors block suppressive effects of progesterone and whether effects of progesterone vary with physiological status. Neither RU 38486 nor RU 43044 blocked the inhibitory effects of progesterone on lymphocyte proliferation. Rather, these antagonists were themselves inhibitory. Effects of progesterone and antagonists were additive: the percentage inhibition caused by progesterone was similar whether antagonists were present or not. The degree of lymphocyte proliferation and the inhibitory effects of progesterone were of the same magnitude for pregnant/lactating cows, pregnant/non-lactating cows, postpartum/lactating cows and cyclic/non-lactating cows. In conclusion, progesterone does not appear to inhibit lymphocyte proliferation through actions that involve classical steroid receptors. There was no evidence that lymphocyte proliferation in the cow is suppressed during pregnancy or that the inhibitory effects of progesterone increase during pregnancy.

Validation of the proximal flow convergence method. Calculation of orifice area in patients with mitral stenosis.

BACKGROUND: It has been proposed recently that measuring the flow convergence region proximal to an orifice by Doppler flow mapping can provide a means of calculating regurgitant flow rate. Although verified in experimental models, this approach is difficult to validate clinically because there is no ideal gold standard for regurgitant flows in patients. However, this method also can be used to derive cardiac output or flow rate proximal to stenotic orifices and therefore to calculate their areas by the continuity equation (area = flow rate/velocity). Applying this method in mitral stenosis would provide a unique way of validating the underlying concept because the predicted areas could be compared with those measured directly by planimetry. METHODS AND RESULTS: We studied 40 patients with mitral stenosis using imaging and Doppler echocardiography. Doppler color flow recordings of mitral inflow were obtained from the apex, and the radius of the proximal flow convergence region was measured at its peak diastolic value from the orifice to the first color alias along the axis of flow. Flow rate was calculated assuming uniform radial flow convergence toward the orifice, modified by a factor that accounted for the inflow funnel angle formed by the mitral leaflets. Mitral valve area was then calculated as peak flow rate divided by peak velocity by continuous-wave Doppler. The calculated areas agreed well with those from three comparative techniques over a range of 0.5 to 2.2 cm2: 1) cross-sectional area by planimetry (y = 1.08x-0.13, r = .91, SEE = 0.21 cm2); 2) area derived from the Doppler pressure half-time (y = 1.02x-0.14, r = .89, SEE = 0.24 cm2); and 3) area calculated by the Gorlin equation in the 26 patients who underwent catheterization (y = 0.89x + 0.08, r = .86, SEE = 0.24 cm2). Agreement with planimetry was similar for 22 patients with mitral regurgitation and 18 without it (P > .6), as well as for 6 in atrial fibrillation (P > .2). CONCLUSIONS: These results validate the proximal flow convergence concept in the clinical setting and also demonstrate that it can be extended to orifice area calculation using the continuity equation.

Malignant lymphomas involving the ovary. A clinicopathologic analysis of 39 cases.

We report 39 patients with non-Hodgkin's lymphoma who initially presented with ovarian enlargement. Fifteen patients had unilateral (10 left, four right, one unknown) and 24 had bilateral ovarian masses. The median size of the tumors was 8 cm (range, 2 to 23 cm). Histologically, using the Working Formulation, 21 lesions (54%) were classified as small noncleaved cell, Burkitt's type; 12 (31%) as large cell (nine diffuse, three focally follicular); three (8%) as diffuse mixed, small and large cell; two (5%) as large cell immunoblastic; and one (2%) as follicular and diffuse small cleaved cell. Twenty-six tumors were analyzed immunophenotypically, 25 (96%) of which were B-cell neoplasms. However, combining histologic and immunohistochemical findings, 37 neoplasms were of B-cell lineage, one diffuse large-cell lymphoma was not analyzed, and one large-cell immunoblastic lymphoma (with features of anaplastic large-cell lymphoma) was of T-cell lineage with an aberrant immunophenotype. On the basis of staging studies and clinical follow-up, we conclude that only four (10%) of the neoplasms in this study most likely arose in the ovary. The primary neoplasms, three diffuse large-cell and one diffuse mixed small- and large-cell, were B-cell neoplasms. Three of four patients with primary neoplasms were apparently cured at last follow-up following surgical excision and chemotherapy. The remainder of the lymphomas in this study, most commonly of small noncleaved cell, Burkitt's type, appear to be systemic tumors that involved the ovaries secondarily. Approximately 40% of patients with systemic neoplasms were alive without evidence of disease at last follow-up.