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PubMed2004

Health care cost implications of sildenafil citrate.

INTRODUCTION: As prescription drug expenditures increase, third-party payors are increasingly scrutinizing costs and reimbursement guidelines, such as for medications that treat chronic conditions that are not necessarily directly life threatening (i.e., obesity, nicotine addiction, and erectile dysfunction). AIM: To review the costs and consequences of pharmacy benefit reimbursement policies related to erectile dysfunction therapies, using sildenafil citrate as the case study. METHODS: Data and references were found through searches of PubMed and Google. RESULTS AND CONCLUSIONS: To meet the varied health needs of a defined population under reasonable resource constraints requires analysis of cost and of the economic and non-economic consequences of a treatment. Based on analysis of sildenafil data, oral therapy of erectile dysfunction provides substantial cost savings when compared with other treatment options and contributes insignificant overall costs to health plans. Regardless, health plans impose controls to limit reimbursement. Because erectile dysfunction is often a precursor to other conditions, such limitations could compromise detection and management of underlying disease (e.g., depressive, cardiovascular, and urogenital). Reimbursement restrictions may also decrease membership satisfaction and re-enrollment rates.

Adult↗
PubMed1979

The effects of trans trans methyl linoleate on the concentration of prostaglandins and their precursors in rat.

Four groups of weanling male rats were fed a diet containing hydrogenated coconut oil (Treatment A); 9-trans, 12-trans linoleate (trans linoleate, (Treatment B); an equal mixture of 9-cis, 12-cis linoleate (cis linoleate) and trans linoleate (Treatment C); and cis linoleate (Treatment D); respectively for 12 weeks. The level of dietary fat was 11% of calories. Only trace amount of eicosatrienoic acid (C20:3w6) was detected in tissues, (liver, platelets) of rats in Treatments A and B. The level of C20:3w6 in platelet lipids of Treatments C and D was 0.1 and 0.33% respectively. The level of arachidonic acid in rats on Treatments A, B, C and D was 2.0, 1.4, 14.1 and 17.6% for platelet lipids, respectively. The serum levels of prostaglandin PGE1 for Treatments A, B, C and D were 1.10 +/- 0.24, 0.22 +/- 0.02, 3.51 +/- 0.58 and 5.69 +/- 0.59 ng/ml, respectively and 2.19 +/- 0.85, 0.15 +/- 0.03, 11.64 +/- 2.63 and 24.89 +/- 4.35 ng/ml for PGE2, respectively. Thus feeding trans linoleate to rats apparently caused decreased biosynthesis of PGs resulting from decreased levels of precursor acids. This was apparently due to the inhibition of conversion of cis linoleate to longer chain polyunsaturated fatty acids. The results indicate that the availability of precursor acids is one limiting factor in PG biosynthesis in rats, and small differences in the level of precursor acids, affected by dietary trans fatty acids may cause large variations in amounts of PGs synthesized.

Animals↗
PubMed1990

Inhibition of murine cytotoxic T lymphocyte activity by a synthetic retroviral peptide and abrogation of this activity by IL.

A synthetic 17 amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins was investigated for its influence on the generation of murine alloantigen-specific CTL activity in vitro. CKS-17 coupled to a carrier protein, BSA or human serum albumin, inhibited the generation of anti-allo CTL in a dose-dependent manner. Controls consisting of BSA and human serum albumin, which had undergone the coupling procedure or neurotensin, an unrelated peptide, coupled to BSA in an identical manner as CKS-17 showed no such inhibitory action. The suppression was not restricted to the Ag specificity of the CTL activity. CKS-17 exerted inhibitory effects on the early afferent phase of CTL induction. Kinetic studies showed that the suppressive activity occurred when CKS-17 was introduced to the immunologically stimulating culture concomitant with or up to 48 h after initiation of culture. Analysis of the frequency of CTL precursor cells using limiting-dilution assays revealed that CKS-17 did act to reduce the number of precursor cells. Abrogation of the inhibition of CTL activity was observed when IL-2 was introduced to the culture together with the stimulator cells. Other lymphokines, such as IL-4, exerted a similar influence to counteract this suppression.

Animals↗
PubMed1985

Limiting dilution analysis of induced unresponsiveness to trinitrophenyl: increased suppression of cytotoxic T precursor cells at unchanged frequencies.

In this paper we address the problem of tolerance in the immune system. We are discussing results of experiments that we designed to distinguish between the two major alternative hypotheses that have been invoked for immunological tolerance: clonal deletion/anergy versus suppression. These alternative hypotheses make essentially different predictions with respect to the frequencies of lymphocytes that react with antigens to which the immune system is tolerant: clonal deletion models predict reduced frequencies, whereas in suppression models precursors of effector cells are postulated to occur at frequencies similar to the nontolerant situation. We investigate these questions by limiting dilution analyses of cytotoxic T cell precursors (CTLP), using two different ways to activate them into functional cytotoxic cells (CTL): polyclonally by Concanavalin A (ConA) and specifically by antigen. Tolerance should be apparent in either protocol since neither antigen nor ConA usually activates self-reactive cytotoxic cells. Moreover, in addition to providing information on precursor frequencies, the multihit results usually obtained in limiting dilution experiments of ConA activated T cells allow the determination of quantitative and qualitative parameters of suppression. In addition, clonal anergy situations could be expected to become apparent by a differential sensitivity of precursors to activation with ConA and with antigen. As experimental system we chose the induced unresponsiveness of mice to trinitrophenyl (TNP) achieved by intravenous injection of reactive trinitrobenzenesulfonic acid (TNBS) and measured in a primary cytotoxic response to TNP-coupled syngeneic cells (TNP-SC). An equally specific but perhaps not identical form of unresponsiveness is induced by coupling the responder cell population with TNBS in vitro. Although we do not propose that this model ideally reflects all aspects of self tolerance, we think that in this particular system of induced unresponsiveness the antigen becomes associated with the surfaces of a large proportion of the cells of the body. Therefore, tolerance to widely expressed cell surface determinants might be quite adequately "simulated" by this experimental unresponsiveness. In our limiting dilution experiments we can distinguish between frequent and infrequent cytotoxic precursors which correspond to virgin and memory cells, respectively. For the frequent CTLP the situation is quite clear: they occur at essentially equal frequencies in normal and tolerant mice, as demonstrated by their reactivity to antigen at low cell densities. However, they are under the influence of a suppressive activity which is strongly increased when compared to that of normal mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Evidence that functional deletion of donor-reactive T lymphocytes in kidney allograft recipients can occur at the level of cytotoxic T cells, IL-2-producing T cells, or both. A limiting dilution study.

The frequencies of circulating donor-reactive cytotoxic lymphocyte precursors (CLP) and Il-2-producing helper lymphocyte precursors (HLP) were determined by limiting dilution analysis in 19 kidney allograft recipients before and at various intervals (up to 2 years) after transplantation. A significant, selective, and stable reduction of the frequencies of donor-reactive (but not of third party-reactive) CLP and/or HLP was observed in some patients beginning 3 to 6 months after transplantation. One patient developed reduced frequency of CLP only, 3 patients reduced frequencies of HLP only, and 2 patients reduced frequencies of both CLP and HLP. The selective reduction of donor-reactive CLP and/or HLP frequencies ranged from 5-25-fold when compared with the pretransplantation level and was associated with stable graft function. These data indicate that functional deletion of circulating donor-reactive T cells can occur at the level of cytotoxic T lymphocytes, Il-2-producing helper T lymphocytes, or both. Implications of these findings for the individualization of immunosuppressive regimens will be discussed.

Humans↗
PubMed2006

Protein splicing in cis and in trans.

Intein-mediated protein splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond. Splice junction proximal residues and internal residues within the intein direct these reactions. The identity of these residues varies in each intein, as groups of related residues populate conserved motifs. Although the basics of the four-step protein splicing pathway are known, mechanistic details are still unknown. Structural and kinetic analyses are beginning to shed some light. Several structures were reported for precursor proteins with mutations in catalytic residues, which stabilize the precursors for crystallographic study. Progress is being made despite limitations inherent in using mutated precursors. However, no uniform mechanism has emerged. Kinetic parameters were determined using conditional trans-splicing (splicing of split precursor fragments after intein reassembly). Several groups concluded that the rate of the initial acyl rearrangement step is rapid and Asn cyclization (step 3) is slow, suggesting that this latter step is rate limiting. Understanding the protein splicing pathway has allowed scientists to harness inteins for numerous applications.

Inteins↗
PubMed2003

Unresponsiveness to lymphoid-mediated signals at the neonatal follicular dendritic cell precursor level contributes to delayed germinal center induction and limitations of neonatal antibody responses to T-dependent antigens.

The factors limiting neonatal and infant IgG Ab responses to T-dependent Ags are only partly known. In this study, we assess how these B cell responses are influenced by the postnatal development of the spleen and lymph node microarchitecture. When BALB/c mice were immunized with alum-adsorbed tetanus toxoid at various stages of their immune development, a major functional maturation step for induction of serum IgG, Ab-secreting cells, and germinal center (GC) responses was identified between the second and the third week of life. This correlated with the development of the follicular dendritic cell (FDC) network, as mature FDC clusters only appeared at 2 wk of age. Adoptive transfer of neonatal splenocytes into adult SCID mice rapidly induced B cell follicles and FDC precursor differentiation into mature FDC, indicating effective recruitment and signaling capacity of neonatal B cells. In contrast, adoptive transfer of adult splenocytes into neonatal SCID mice induced primary B cell follicles without any differentiation of mature FDC and failed to correct limitations of tetanus toxoid-induced GC. Thus, unresponsiveness to lymphoid-mediated signals at the level of neonatal FDC precursors delays FDC maturation and GC induction, thus limiting primary Ab-secreting cell responses to T-dependent Ags in early postnatal life.

Aging↗
PubMed1996

The weak CD8+ CTL response to an influenza hemagglutinin epitope reflects limited T cell availability.

One of the two class I MHC (H-2Kd)-restricted immunogenic regions identified on the influenza virus strain A/Japan/57 (H2N2) hemagglutinin (HA) encompasses two distinct, partially overlapping epitopes. These epitopes map to residues 204 to 212 (JHA 204-212) and 210 to 219 (JHA 210-219), respectively. When we investigated the magnitude of the CTL responses of H-2d BALB/c mice to these two epitopes, we found that the JHA 204-212 nonamer epitope is immunodominant, eliciting vigorous CTL responses in BALB/c mice immunized with A/Japan/57 virus. In contrast, the CTL response to the JHA 210-219 decamer epitope was weak and variable. The subdominance of the JHA 210-219 was not due to low affinity binding of JHA 210-219 to H-2Kd or to inefficient processing of this epitope in vivo. Rather, an analysis of CTL precursor (pCTL) frequency by limiting dilution showed that the frequency of pCTL to the JHA 210-219 epitope was at least 10-fold lower than the frequency of pCTL to the JHA 204-212 epitope, implying that the low and variable response to the JHA 210-219 epitope was due to a limited number of CD8+ T cell precursors directed to JHA 210-219. This hypothesis was further supported by the finding of limited heterogeneity in reactivity pattern displayed by short term bulk cultures of the JHA 210-219-specific CTLs for cross-reactive epitopes. Implications of these findings for vaccine design and for T lymphocyte function and repertoire development are discussed.

Amino Acid Sequence↗
PubMed1989

The processing of a cathepsin L precursor in vitro.

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.

Animals↗
PubMed1987

Conformation limited proteolysis in the common neurophysin-copeptin precursor shown by trypsin-sepharose chromatographic proteolysis.

The guinea pig two-domain precursor of MSEL-neurophysin and copeptin has been passed through a trypsin-Sepharose column in order to mimic the enzyme processing by a membrane-bound endopeptidase. Only two cleavages were observed located in the inter-domain sequence (at Arg-94 and Arg-98), in contrast to several additional cleavages found when free neurophysin or copeptin is subjected to soluble trypsin. Because the physiological maturation involves a single cleavage at Arg-94, both local accessibility in the precursor and narrow specificity of the enzyme are implied in the processing.

Amino Acid Sequence↗
PubMed1984

Limitations of Zilversmit approach to analysis of precursor-product relationships.

Recently, a modification of the Zilversmit method has been used to establish the extent to which a precursor-product relationship exists between the phospholipids of the lamellar bodies (PLlb) and those in the alveolar compartment (PLalv) of the rabbit lung. According to this analysis, if a plot of specific activity (sp act) of PLalv vs. the integral with respect to time of (sp act PLlb-sp act PLalv) results in a straight line, then a presumptive precursor-product relationship exists; the slope of this line will indicate the turnover time of the product compartment. We have established analytically and confirmed numerically that collinearity is, in fact, a poor test of precursor-product relationship. We examine some causes of hysteresis and the direction of hysteresis of this plot for compartmental configurations other than precursor product. Finally, we apply the conclusions of our analysis to some of the published data relating to the relationship between PLlb and PLalv, and we indicate where previous conclusions may now be considered premature.

Body Fluid Compartments↗
PubMed2004

CD4-directed peptide vaccination augments an antitumor response, but efficacy is limited by the number of CD8+ T cell precursors.

Peptide vaccination is an immunotherapeutic strategy being pursued as a method of enhancing Ag-specific antitumor responses. To date, most studies have focused on the use of MHC class I-restricted peptides, and have not shown a correlation between Ag-specific CD8(+) T cell expansion and the generation of protective immune responses. We investigated the effects of CD4-directed peptide vaccination on the ability of CD8(+) T cells to mount protective antitumor responses in the DUC18/CMS5 tumor model system. To accomplish this, we extended the amino acid sequence of the known MHC class I-restricted DUC18 rejection epitope from CMS5 to allow binding to MHC class II molecules. Immunization with this peptide (tumor-derived extracellular signal-regulated kinase-II (tERK-II)) induced Ag-specific CD4(+) T cell effector function, but did not directly prime CD8(+) T cells. Approximately 31% of BALB/c mice immunized with tERK-II were protected from subsequent tumor challenge in a CD40-dependent manner. Priming of endogenous CD8(+) T cells in immunized mice was detected only after CMS5 challenge. Heightened CD4(+) Th cell function in response to tERK II vaccination allowed a 12-fold reduction in the number of adoptively transferred CD8(+) DUC18 T cells needed to protect recipients against tumor challenge as compared with previous studies using unimmunized mice. Furthermore, tERK-II immunization led to a more rapid and transient expansion of transferred DUC18 T cells than was seen in unimmunized mice. These findings illustrate that CD4-directed peptide vaccination augments antitumor immunity, but that the number of tumor-specific precursor CD8(+) T cells will ultimately dictate the success of immunotherapy.

Adjuvants, Immunologic↗
PubMed1991

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of arylsulfatase B.

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease.

Alleles↗
PubMed1983

Role of whole-body lipids and nitrogen as limiting factors for survival in tumor-bearing mice with anorexia and cachexia.

This study was designed to ascertain whether the overall availability of whole-body lipids and nitrogen is a limiting factor for survival in tumor-bearing mice suffering from anorexia and cachexia. Three-month-old nongrowing mice (C57BL/6J) were given s.c. transplants of a methylcholanthrene-induced sarcoma. Freely fed, starved, and pair-fed animals were used. Body and lipid composition, tumor growth, and survival time were measured. Freely fed sarcoma-bearing mice died with profoundly altered body composition. This was not explained by the anorexia assessed in pair-feeding experiments. Starvation had caused a more severe depletion in body composition in both tumor-bearing and nontumor-bearing animals than the tumor alone did in freely fed tumor-bearing mice. Freely fed tumor-bearing animals had normal proportions of whole-body triglycerides, cholesterol, and polar lipids, but they lost palmitic acid quantitatively more than any other fatty acid. It is unlikely that any single fatty acid became limiting during tumor growth. The results show that the overall availability of lipids, nitrogen, and glucose precursors is not a limiting factor for survival in experimental tumor cachexia. Other factors considered to be more likely as determining factors for the death of tumor-bearing animals are discussed.

Animals↗
PubMed1993

Differentiation of neurogenic precursors within the neural crest cell lineage.

It has been suggested that many, if not all crest-derived neurons develop from a limited subpopulation of neurogenic precursors. To develop cell-type specific markers that identify these precursors directly we have used differential screening of crest-derived cell populations known to have, or not to have, neurogenic ability. We have determined that the neuron-specific human auto-antibodies designated Anti-Hu bind to cytoplasmic and nuclear determinants not only in mature avian neurons and neuroendocrine cells but also in subpopulations of morphologically non-neuronal avian crest-derived cells. Significantly, these Anti-Hu+ non-neuronal crest-derived cells are present only in populations that have neurogenic ability and are absent from populations that lack neurogenic ability. Moreover, following additional development in vivo or in vitro, Anti-Hu+ non-neuronal crest-derived cells appear to express other neuronal traits. These results suggest that Anti-Hu-immunoreactivity is an early indicator of neurogenesis among crest-derived cells, and that Anti-Hu+ non-neuronal cells are either neurogenic precursors or immature neurons. Similarly, using the same differential screening paradigm, we have identified two monoclonal antibodies, designated 12E10 and 17F5, which also label both neurons and some apparently nonneuronal cells in neurogenic populations of neural crest cells. Anti-Hu-IR appears to precede expression of either of these two markers.

Animals↗
PubMed2003

Aged mice exhibit distinct B cell precursor phenotypes differing in activation, proliferation and apoptosis.

Senescence in murine models is associated with a reduction, albeit heterogeneous, in bone marrow pre-B cells. We have categorized aged BALB/c mice into two phenotypes based on their patterns of pre-B/pro-B cell loss. Each phenotype is characterized by distinct responses to the growth cytokine IL-7 and capacity for survival in vitro. A 'moderate' loss of late-stage pre-B cells (25-80%) coincided with decline in proliferation to rmIL-7. This was also associated with a decrease in the frequency of pro-B cells which increased phosphotyrosine content upon IL-7 stimulation, an indicator of early activation events. A 'severe' loss of pre-B cells (>80%) resulted in a reduced pro-B cell pool which retained normal activation and proliferative responses to IL-7. B cell precursors from aged mice with severe alterations in B lymphopoiesis displayed increased susceptibility to apoptosis in comparison to both aged mice with moderate B cell precursor loss and young mice. Conceivably, during senescence, aged mice may initially accumulate B cell precursors which are poorly responsive to IL-7. Progressively, these refractory B cell precursors may be eliminated via apoptosis; however, the remaining limited pool of B cell precursors retains the capacity to respond to IL-7 stimulation.

Aging↗
PubMed1995

Overexpression of the protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), in human epithelial ovarian cancer.

Our current understanding of human ovarian tumorigenesis is limited by the lack of a discrete precursor lesion as well as a limited knowledge of the steps in tumor progression. Since the alterations in the regulation of the tyrosyl residues on various cellular proteins appear to be an important pathway in neoplastic transformation, it is possible that changes in the expression of the proteins that control tyrosine phosphorylation (i.e., tyrosine kinases and phosphatases) may play a role in ovarian cancer development. Protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), contains two src homology 2 domains and is expressed primarily in hematopoietic and epithelial cells. Using Northern blot and immunoblotting analysis, we showed that both the PTPN6 transcripts and proteins were overexpressed two- to four-fold in 7 of the 8 ovarian epithelial carcinoma cell lines studied. In addition, we showed that there was also a two- to threefold increase in expression of the PTPN6 transcript in 10 of 11 (91%) invasive ovarian epithelial cancer tissues examined. These observations suggest that the PTPN6 gene is potentially of etiologic relevance to a majority of ovarian cancers.

Epithelium↗
PubMed1995

17 beta-Hydroxysteroid dehydrogenase activity in endometrial cancer cells: different metabolic pathways of estradiol in hormone-responsive and non-responsive intact cells.

In this paper we report that two human long-term endometrial cancer cell lines, Ishikawa and HEC-1A, exhibit quite different abilities in metabolizing estrogens. As a matter of fact, incubation of Ishikawa cells with close-to-physiological concentrations of estradiol (E2) as precursor resulted in: (1) elevated formation (up to 90%) of E2-sulphate (E2-S), using lower precursor concentrations; (2) very limited conversion to estrone (E1) (< 10% at 24 h incubation), as either free or sulphate; and (3) low but consistent production of other estrogen derivatives, such as 2-hydroxy-estrogens and estriol. Conversely, scant amounts (if any) of E2-S were found in HEC-1A cells, while no detectable formation of other estrogen metabolites could be observed after 24 h. On the other hand, E1 production was significantly greater (nearly 60% at 24 h) than in Ishikawa cells, a large proportion of E1 (over 50% of the total) being formed after only 6 h incubation using time-course experiments. The hypothesis that E2 metabolism could be minor in Ishikawa cells as a consequence of the high rate of E2-S formation encountered is contradicted by the evidence that conversion to E1 also remains limited in the presence of much lower E2-S amounts, seen using higher molar concentrations of precursor. Overall, we observe that 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity diverges significantly in intact Ishikawa and HEC-1A endometrial cancer cells. This difference could not merely be accounted for by the diverse amounts of substrate (E2) available to the cells, nor may it be imputed to different levels of endogenous estrogens. It should rather be sought in different mechanisms controlling 17 beta-HSD activity or, alternatively, in the presence of distinct isoenzymes in the two different cell types.

Cell Division↗