Pharmacological study of anti-inflammatory activity of aqueous extracts of Mikania glomerata (Spreng.) and Mikania laevigata (Sch. Bip. ex Baker).

ETHNOPHARMACOLOGICAL RELEVANCE: Mikania glomerata Spreng. (MG) and Mikania laevigata Sch. Bip. ex Baker (ML), popularly known as guaco, are medicinal plants similar in morphology, chemical composition and medicinal uses. Both species are often used and sold without distinction; however, it is believed that their chemical composition is different. AIM: Thus, the aim of this study is to investigate if the aqueous extract of MG and ML present similar anti-inflammatory activity to the point of being used interchangeably. MATERIAL AND METHODS: Different doses of both extracts and coumarin were given to rats in different experimental models to assess the anti-inflammatory activity between these two species. For this, the animals were submitted to paw edema, pleurisy and degranulation of peritoneal mast cell and the extracts were also characterized by Ultra High Efficiency Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS). RESULTS: The chromatographic method showed that ML presents ten times more coumarin than MG. Oral administration of MG, ML and coumarin inhibited paw edema induced by carrageenan (400 mg/kg, 55% inhibition; 400 mg/kg, 57% inhibition; 75 mg/kg, 38% inhibition; p < 0.05, respectively). MG, ML and coumarin treatment also inhibited the edema induced by compound 48/80 (400 mg/kg, 56% inhibition; 400 mg/kg, 69% inhibition; 75 mg/kg, 40% inhibition; p < 0.05, respectively). MG, ML and coumarin did not prevent mast cell degranulation and the consequent histamine release in Wistar rat peritoneal mast cells induced by compound 48/80. MG did not inhibit cell infiltration in pleurisy nor the highest dose tested, while ML decreased the leukocyte migration (200 and 400 mg/kg, 23% and 30% inhibition; p < 0.001, respectively) and, to a lesser extent, coumarin also reduced cell infiltration (10, 50 and 75 mg/kg; 15%, 16% and 17% inhibition; p < 0.001, respectively). CONCLUSION: The variation of the results of the anti-inflammatory activity found in M. glomerata and M. laevigata demonstrates that these two species should not be used interchangeably. Coumarin, as already proven, has anti-inflammatory action however, we have suggested that it probably is not the only component responsible for this therapeutic effect in the extracts.

Clinical significance of heparin-binding epidermal growth factor-like growth factor in peritoneal fluid of ovarian cancer.

Epidermal growth factor receptor (EGFR) has been implicated in tumour growth and extension of ovarian cancer. Peritoneal fluid in ovarian cancer patients contains various growth factors that can promote tumour growth and extension. In order to investigate the clinical significance of EGFR ligands as activating factors of ovarian cancer, we examined the cell proliferation-promoting activity and the level of EGFR ligands in peritoneal fluid obtained from 99 patients. Proliferation-promoting activity in peritoneal fluid from 63 ovarian cancer patients (OVCA) was much higher than peritoneal fluid from 18 ovarian cyst patients (OVC) and 18 normal ovary patients (NO), and the activity was suppressed only by antibodies against EGFR or heparin-binding epidermal growth factor (HB-EGF). A large difference was observed in the level of EGFR ligands between HB-EGF and TGF-alpha or amphiregulin. The concentration of HB-EGF in OVCA significantly increased compared to that in OVC or NO (P<0.01). No significant difference in the concentration of TGF-alpha and amphiregulin was found between the OVCA and NO or OVC groups. In peritoneal fluid, HB-EGF is sufficiently elevated to activate cancer cells even at an early stage of OVCA. These results suggested that HB-EGF in peritoneal fluid might play a key role in cell survival and in the proliferation of OVCA.

Atropine prevents midazolam-induced core hypothermia in elderly patients.

STUDY OBJECTIVE: To test the hypothesis that core temperature is well preserved when atropine and midazolam are combined. DESIGN: Randomized, blinded study. SETTING: Department of Anesthesia, Yamanashi Medical University. PATIENTS: 40 elderly, ASA physical status I and II patients (aged more than 60 years). INTERVENTIONS: Patients were randomly assigned (n = 10 per group) to premedication with: 1) saline control; 2) midazolam 0.05 mg/kg; 3) atropine 0.01 mg/kg; and 4) midazolam 0.05 mg/kg combined with atropine 0.01 mg/kg. All premedication was given on the ward at approximately 8:30 am, approximately 30 minutes before induction of anesthesia. MEASUREMENTS AND MAIN RESULTS: Core temperatures were measured at the right tympanic membrane. Mean skin temperature was calculated as 0.3 x (T(chest) + T(arm)) + 0.2 x (T(thigh) + T(calf)). Fingertip perfusion was evaluated using forearm minus fingertip and calf minus toe, skin-surface temperature gradients. Temperatures were evaluated at the time of premedication and 30 minutes later, just before induction of anesthesia. Core temperature remained nearly constant in the control patients (0.1 +/- 0.2 degrees C; mean +/- SD), whereas it decreased significantly in the patients given midazolam alone (-0.3 +/- 0.1 degrees C). Atropine alone increased core temperature (0.3 +/- 0.2 degrees C), although the increase was not statistically significant. The combination of midazolam and atropine attenuated the hypothermia induced by midazolam alone (0.0 +/- 0.2 degrees C). Initial skin-temperature gradients exceeded 0 degrees C in all groups, indicating that the patients were vasoconstricted. The gradients were unchanged by premedication with saline or atropine. Midazolam significantly decreased the gradient (-1.8 +/- 1.1 degrees C), as did the combination of midazolam and atropine (-1.4 +/- 0.9 degrees C). CONCLUSIONS: The thermoregulatory effects of benzodiazepine receptor agonist and cholinergic inhibitors oppose each other, and the combination leaves core temperature unchanged.

Radiation therapy for prostate cancer.

OBJECTIVES: To review the advances in radiation therapy for prostate cancer and the nursing care of patients with prostate cancer. DATA SOURCES: Peer-reviewed journal articles, including research studies and review articles. CONCLUSIONS: Radiation therapy is used to cure early stage prostate cancer, control locally advanced disease, and effectively palliate symptoms of metastasis. The three forms of treatment used include external beam radiation therapy, brachytherapy; and radiopharmaceutical treatments. IMPLICATIONS FOR NURSING PRACTICE: Nursing care of patients receiving radiation therapy for prostate cancer includes managing the symptoms associated with the disease and treatment, educating patients and families about self-care measures, and providing support throughout the course of the disease.

Identification of serum factor inducing ectodomain shedding of proHB-EGF and sStudies of noncleavable mutants of proHB-EGF.

The ectodomain of the transmembrane form of HB-EGF (proHB-EGF) is cleaved at the cell surface by proteases, yielding a soluble growth factor. A number of stimuli, including TPA, accelerate this cleavage. However, proHB-EGF is shed constitutively under normal culture conditions without any particular stimuli. We demonstrate here that constitutive cleavage resulted largely from factor(s) contained in supplemented FCS in a culture medium. Analysis of serum factors, including digestion with enzymes, separation by thin layer chromatography, and shedding assay with purified phospholipids, revealed that lysophosphatidic acid (LPA) is a major factor in FCS for stimulation of proHB-EGF shedding. We also studied here ectodomain shedding of two kinds of mutant form of proHB-EGF which have a single amino acid substitution around the putative cleavage sites. These mutant forms showed resistance to stimuli of both TPA and LPA, suggesting that proHB-EGF is cleaved at the similar site by stimulation with TPA and LPA.

Immunohistochemical distribution of CD9, heparin binding epidermal growth factor-like growth factor, and integrin alpha3beta1 in normal human tissues.

The tetra-membrane-spanning protein CD9 forms a complex with a membrane-anchored heparin binding epidermal growth factor-like growth factor (HB-EGF) and integrin alpha3beta1 in some human and monkey cell lines. We show here the immunohistochemical distribution of CD9, HB-EGF, and integrin alpha3beta1 in normal human tissues. Distribution of CD9, HB-EGF, and integrin alpha3beta1 was similar in various tissues, including transitional epithelium, squamous epithelium, thyroid follicular epithelium, adrenal cortex, testis, smooth muscle, and stromal fibrous tissue. However, distribution of the three proteins did not coincide in some tissues, such as lung, liver, kidney, gastric and intestinal epithelium, pancreas, salivary gland, and ovary. In striated muscle, including cardiac muscle, CD9 was present not in the muscle cells themselves but in the endomysium and perimysium, whereas HB-EGF was distributed in the muscle cells themselves. CD9 was distributed in the myelin, but HB-EGF was found in the axon of the peripheral and central nervous systems. Coincident distribution of integrin alpha3beta1 with others was not observed in muscles and neural tissues. In conclusion, there is a possibility of complex formation and functional cooperation of CD9 with HB-EGF and/or integrin alpha3beta1 in several tissues.

Heparin-binding EGF-like growth factor: a juxtacrine growth factor.

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family growth factors, is synthesized as a membrane-anchored form (proHB-EGF). Proteolytic cleavage of proHB-EGF at the extracellular domain yields the soluble form of HB-EGF (sHB-EGF). ProHB-EGF is not only the precursor molecule for sHB-EGF but also a biologically active molecule itself. Recent studies indicate that proHB-EGF has unique properties distinct from the soluble form. ProHB-EGF forms a complex with membrane proteins including a tetramembrane spanning protein: CD9, an adhesion molecule integrin: alpha3beta1, and heparan sulfate proteoglycans. The complex is localized at the cell-cell contact site, suggesting that proHB-EGF may function in cell-to-cell signaling by a juxtacrine mechanism. In an in vitro model system, proHB-EGF showed growth inhibitory activity, while sHB-EGF was growth stimulatory. Ectodomain shedding, conversion of the membrane-anchored form into the soluble form, is regulated by multiple signaling pathways. All these characteristics imply that proHB-EGF and sHB-EGF are used in different ways. In vivo functions of sHB-EGF and proHB-EGF have been largely undefined, but recent studies implicate them in a variety of physiological processes including blastocyst implantation and wound healing.

Identification of mammalian TOM22 as a subunit of the preprotein translocase of the mitochondrial outer membrane.

A mitochondrial outer membrane protein of approximately 22 kDa (1C9-2) was purified from Vero cells assessing immunoreactivity with a monoclonal antibody, and the cDNA was cloned based on the partial amino acid sequence of the trypsin-digested fragments. 1C9-2 had 19-20% sequence identity to fungal Tom22, a component of the preprotein translocase of the outer membrane (the TOM complex) with receptor and organizer functions. Despite such a low sequence identity, both shared a remarkable structural similarity in the hydrophobicity profile, membrane topology in the Ncyt-Cin orientation through a transmembrane domain in the middle of the molecule, and the abundant acidic amino acid residues in the N-terminal domain. The antibodies against 1C9-2 inhibited the import of a matrix-targeted preprotein into isolated mitochondria. Blue native polyacrylamide gel electrophoresis of digitonin-solubilized outer membranes revealed that 1C9-2 is firmly associated with TOM40 in the approximately 400-kDa complex, with a size and composition similar to those of the fungal TOM core complex. Furthermore, 1C9-2 complemented the defects of growth and mitochondrial protein import in Deltatom22 yeast cells. Taken together, these results demonstrate that 1C9-2 is a functional homologue of fungal Tom22 and functions as a component of the TOM complex.

Involvement of deregulated epiregulin expression in tumorigenesis in vivo through activated Ki-Ras signaling pathway in human colon cancer cells.

To identify the genes located downstream of the activated Ki-Ras signaling pathways in human colon cancer cells, a PCR-based cDNA subtraction library was constructed between HCT116 cells and HCT116-derived activated Ki-ras-disrupted cells (HKe3). One of the genes in HCT116 that was evidently up-regulated was epiregulin, a member of the epidermal growth factor family that is expressed in many kinds of human cancer cells. HKe3-stable transfectants expressing activated Ki-Ras regained over-expression of epiregulin. To further elucidate the biochemical structure and significance of epiregulin expression in tumorigenesis, HKe3-stable transfectants expressing epiregulin (e3-pSE cells) were established. Epiregulin existed as highly glycosylated membrane-bound forms, and TPA rapidly induced ectodomain shedding of epiregulin. Furthermore, the conditioned medium of e3-pSE cells showed more DNA synthesis for 32D cells expressing epidermal growth factor receptor (DER) cells than that of HKe3. Although anchorage-independent growth in soft agar was not observed for e3-pSE cells, tumorigenicity in nude mice was observed evidently, and their growth rate was correlated with each amount of exogenous epiregulin expression. These results suggested that activated Ki-Ras will be one of the factors contributing to the overexpression of epiregulin in human colon cancer cells, and that epiregulin will play a critical role in human tumorigenesis in vivo.

Contact-dependent growth inhibition and apoptosis of epidermal growth factor (EGF) receptor-expressing cells by the membrane-anchored form of heparin-binding EGF-like growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) transduces mitogenic signals through the EGF receptor (EGFR). There are two forms of HB-EGF, the membrane-anchored form (pro-HB-EGF) and the soluble form (sHB-EGF). We studied the biological activity of pro-HB-EGF by using a model in which pro-HB-EGF-expressing effector cells was co-cultured with EGFR-expressing target cells. The DER cell, an EGFR-expressing derivative of the interleukin-3-dependent hematopoietic 32D cell line, grows well in the presence of EGF or sHB-EGF without IL-3. When DER cells were co-cultured on a monolayer of Vero-H cells overexpressing pro-HB-EGF, growth inhibition and subsequent apoptosis were induced in the DER cells even in the presence of excess amounts of EGF or sHB-EGF. Such growth inhibition of DER cells was abrogated when specific antagonists for pro-HB-EGF were added in the culture medium or when direct contact of DER cells with Vero-H cells was prevented, indicating that pro-HB-EGF is involved in this inhibitory effect. Pro-HB-EGF-induced apoptosis of DER cells was also observed even in the presence of IL-3. This rules out the possibility of simple competition between soluble EGFR ligands and pro-HB-EGF. Moreover, 32D cells expressing EGFR mutant composed of the extracellular and the transmembrane domain of EGFR and the cytoplasmic domain of erythropoietin receptor did not undergo apoptosis by co-culture with Vero-H cells, indicating that the inhibitory signal induced by pro-HB-EGF-expressing Vero-H cells is mediated to DER cells via EGFR and that the cytoplasmic domain of EGFR is essential for pro-HB-EGF-induced apoptosis. From these results, we concluded that pro-HB-EGF has unique biological activity through cell-cell contact that is distinct from the activity of sHB-EGF.

Purification and characterization of D-glucosaminitol dehydrogenase from Agrobacterium radiobacter.

D-Glucosaminitol dehydrogenase, which catalyzes the conversion of D-glucosaminitol to 3-keto-D-glucosaminitol, was purified to apparent homogeneity from extracts of Agrobacterium radiobacter. This organism has constitutively depressed levels of the enzyme but expression of the enzyme is induced by addition of D-glucosamine to the medium. Purification included ammonium sulfate fractionation and chromatography on columns of DEAE-Sephacel, Octyl-Sepharose CL-4B, and Cellulofine. The purified enzyme migrated as a single band, coinciding with dehydrogenase activities specific for D-glucosaminitol and ethanol, when electrophoresed on a 7.5% polyacrylamide gel at pH 8.0. Electrophoresis on a 12.5% PAGE in the presence of 1% SDS also yielded a single band. The enzyme had an apparent molecular mass of 79 kDa, as measured by the pattern of elution from a column of Cellulofine. The results indicated that the enzyme was a dimer of identical (or nearly identical) subunits of 39.5 kDa. D-Glucosaminitol dehydrogenase required NAD+ as a cofactor and used ethanol as the preferred substrate, as well as aliphatic alcohols with 2 to 4 carbon atoms, D-glucosaminitol, D-glucosaminate, DL-allothreonine, glycerol, and erythritol as additional substrates. In 50 mM Tris-HCl buffer (pH 9.0) at 25 degrees C, the K(m) for D-glucosaminitol, ethanol, and NAD+ were 2.2, 2.0, and 0.08 mM, respectively. The enzyme had a pH optimum of 10 for D-glucosaminitol and 8.5 for ethanol. The enzyme lost substantial activity when treated with pyrazole, with certain reagents that react with sulfhydryl groups and with Zn2+ ion. The various results together suggest that the enzyme exploits different amino acid residues for the dehydrogenation of ethanol and of D-glucosaminitol.

A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.

Enhancement of DNA-transfection frequency by X-rays.

This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy.

Expression and distribution of CD9 in myelin of the central and peripheral nervous systems.

CD9 is a member of the newly identified tetra-membrane-spanning protein family. We show here that CD9 is a constituent of myelin in the central and peripheral nervous systems. Expression of CD9 was detected in human cerebral white matter and sciatic nerve by Northern and Western blotting. Myelin in the central and peripheral nervous systems was strongly stained with a monoclonal antibody against human CD9 antigen in paraffin-embedded sections. CD9 was detected in adult nervous tissue but not in developing brain at less than 20 weeks of gestation. Immunohistochemical studies indicated that expression of CD9 is correlated with myelination and is somewhat delayed compared with expression of myelin basic protein, a major component protein of myelin. In the central nervous system, CD9 was detected along the outermost membrane of compact myelin but not inside compact myelin or the periaxonal region. Although the membrane-anchored form of heparin-binding epidermal-growth-factor-like growth factor (proHB-EGF), which is identical to the diphtheria toxin receptor, forms a complex with CD9 in some human and monkey cell lines, proHB-EGF was not detected in myelin immunocytochemically. The distribution of CD9 in the outer surface of myelin and its relatively late developmental appearance suggest that CD9 may interact with the extracellular matrix or cell adhesion molecules and participate in the maintenance of the entire myelin sheath.

A nursing perspective on radiation-induced xerostomia.

Xerostomia during and following a course of head and neck irradiation profoundly impacts the quality of life of many patients. Xerostomia not only affects mucous membranes and teeth but also interferes with patient comfort, nutrition, and activities of daily living. A thorough evaluation of xerostomia is essential and should include providing anticipatory guidance to the patient and family. In addition, education on prophylactic oral care is necessary during the initial phases of treatment. As symptoms occur, various palliative interventions are tailored to the patient's and family's needs, promoting adherence to mouth care regimens and enhancing patient comfort. Long-term follow-up with education and counseling is critical for optimal patient management.

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) and diphtheria toxin receptor-associated protein (DRAP27)/CD9 form a complex with integrin alpha 3 beta 1 at cell-cell contact sites.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors, which interact with EGF receptor to exert mitogenic activity. The membrane-anchored form of HB-EGF, proHB-EGF, is biologically active, providing mitogenic stimulation to neighboring cells in a juxtacrine mode. ProHB-EGF forms a complex with diphtheria toxin receptor-associated protein (DRAP27)/CD9, a tetra membrane-spanning protein that upregulates the juxtacrine mitogenic activity of proHB-EGF. We explored whether other proteins associate with DRAP27/CD9 and proHB-EGF. Immunoprecipitation with anti-DRAP27/CD9 resulted in preferential coprecipitation of integrin alpha 3 beta 1 from Vero cell, A431 cell and MG63 cell lysates. Anti-integrin alpha 3 or anti-integrin beta 1 coprecipitated DRAP27/CD9 from the same cell lysates. Chemical cross-linking confirmed the physical association of DRAP27/CD9 and integrin alpha 3 beta 1. Using Vero-H cells, which overexpress HB-EGF, we also demonstrated the association of proHB-EGF with DRAP27/CD9 and integrin alpha 3 beta 1. Moreover, colocalization of proHB-EGF, DRAP27/CD9, and integrin alpha 3 beta 1 at cell-cell contact sites was observed by double-immunofluorescence staining. At cell-cell contact sites, DRAP27/CD9 was highly coincident with alpha-catenin and vinculin, suggesting that DRAP27/CD9, proHB-EGF, and integrin alpha 3 beta 1 are colocalized with adherence junction-locating proteins. These results indicate that direct interaction of growth factors and cell adhesion molecules may control cell proliferation during the cell-cell adhesion process.

D-glucosaminate aldolase activity of D-glucosaminate dehydratase from Pseudomonas fluorescens and its requirement for Mn2+ ion.

When D-glucosaminate dehydratase (GADH) was incubated with D-glucosaminate (GlcNA) in veronal buffer (VB; 0.01 M, pH 8.0), GlcNA was converted stoichiometrically to glyceraldehyde, pyruvate, and ammonia (aldolase reaction A). This reaction occurred in addition to the dehydratase reaction (conversion of GlcNA to 2-keto-3-deoxy-D-gluconate and ammonia: alpha,beta-elimination reaction, B). The ratio of the activities (A:B) was about 1:4. However, in potassium phosphate buffer (KPB; 0.04 M, pH 8.0), the aldolase reaction was inhibited to 3-4% of that in VB, and also inhibited by various derivatives of glycerol, in particular, glycerol-3-phosphate (glycerol-3-P) and glyceraldehyde-3-phosphate (glyceraldehyde-3-P) in VB. The native enzyme was inhibited by incubation with 0.1 M EDTA, and the activity was restored by incubation of the EDTA-treated enzyme with (Mn2+ + pyridoxal 5'-phosphate (PLP)). When the EDTA-treated enzyme was incubated with (Mn2+ + PLP + glycerol-3-P), the activity of reaction B increased to 131% but that of reaction A decreased to 21%. These results suggested that Mn2+, PLP, and the phosphate group of glycerol-3-P are involved in formation of the active enzyme. In the case of the aldolase reaction, Mn2+ ion, which might be essential for the reaction, is chelated by the phosphate group of glycerol-3-P with resultant inhibition of the aldolase reaction.

The membrane protein CD9/DRAP 27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin-binding EGF-like growth factor.

The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CD9/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.

Oral pilocarpine for radiation-induced xerostomia: integrated efficacy and safety results from two prospective randomized clinical trials.

PURPOSE: Pilocarpine hydrochloride administered in either a fixed-dose or in a dose-titration protocol three times a day for 12 weeks was evaluated for its ability to relieve symptoms of postradiation xerostomia and to improve saliva production. The studies were randomized, double-blind, placebo-controlled, multicenter clinical trials. A total of 369 patients who had received at least 40 Gy of radiation to the head and neck with clinically significant xerostomia were enrolled in the two studies. In the dose-titration study, 162 patients were enrolled and they received a thrice daily regimen of 2.5 mg tablets for first 4 weeks, 5.0 mg tablets for the second 4 weeks, and 10.0 mg tablets for last 4 weeks of a 12-week study. Patients in the titration study were allowed to down titrate following at least one dose escalation to alleviate bothersome side effects, if any. In the fixed dose study, 207 patients received either placebo, 5.0 mg, or 10.0 mg tablets t.i.d. for 12 weeks. METHODS AND MATERIALS: Patients were evaluated for symptomatic relief by responding to questionnaires using visual analog scales and categorical questions; and, for saliva production by sialometry. Questionnaires measured relief of intraoral dryness, improvement in overall condition (global response), oral discomfort, difficulty in speaking, chewing and swallowing, denture wearing, and usage of artificial saliva. Evaluations were conducted at baseline, and weeks 4, 8, and 12. RESULTS: There were statistically significant improvements in salivary flow in pilocarpine treatment groups vs. placebo. There was a significant improvement in the overall "global" condition of xerostomia associated with the use of pilocarpine in both studies. In the fixed-dose study, there were significant improvements in oral dryness, mouth comfort, ability to speak, and reduction in the use of oral comfort agents. The dose-titration study showed improvements in dryness that approached significance (p = 0.057) and a decreased use of oral comfort agents (p = 0.045). All pilocarpine dosages (2.5, 5.0, and 10.0 mg three times a day) were judged to be safe. Adverse experiences were those expected for a cholinergic agonist, with the most common being mild to moderate sweating. The incidence of these events increased by dose. CONCLUSION: It is concluded that in these studies pilocarpine produced clinically significant benefits with acceptable side effects and risks for the treatment of symptomatic postradiation xerostomia. The incidence of most adverse events increased with dose. Best results may require continuous treatment for more than 8 weeks with doses greater than 2.5 mg three times a day. A 5.0 mg thrice daily regimen produced the best clinical results when both efficacy and side effects were taken into consideration. There may be some patients who would experience some additional benefit by increasing the dose to 10 mg thrice daily.