[Concurrent chemoradiation therapy with nedaplatin for high-risk cervical cancer--clinical investigation of adverse events].

A clinical investigation of adverse events was conducted to confirm the safety of concurrent chemotherapy using nedaplatin (cisplatin derivative) and radiotherapy in the high-risk carcinoma of the uterine cervix. Seven patients who were treated with radical radiotherapy and 5 patients who were treated with adjunctive radiotherapy after radical hysterectomy and pelvic lymphadenectomy were eligible for the study. Nedaplatin was given intravenously at 70 mg/m2 on day 1 and day 29, and a total of 24 courses of nedaplatin administration were observed. None of the planned radiotherapy was postponed or discontinued due to side effects. Major adverse effects observed were gastrointestinal effects such as anorexia (66.7%), nausea and vomiting (33.3%) and diarrhea (66.7%). Grade 3 (in the 2nd course) and Grade 4 (in the 1st course) diarrhea was observed in one patient, which was easily relieved by antidiarrheal. Hematologic side effects were also major, including leukopenia (62.5%), neutropenia (75.0%), anemia (75.0%), and thrombocytopenia (33.3%). Hematologic effects were generally moderate; no Grade 4 (severe) effects were observed. Although these hematologic effects were lasting longer compared with radiation therapy alone, there were no significant differences in the seriousness of these side effects. Concurrent chemoradiation therapy with nedaplatin 70 mg/m2 every 4 weeks was safe and adverse effects were self-limited or resolved with medical management. Dose escalation in the phase III clinical study may be considered.

[Vascular anastomoses in free jejunal reconstruction to the neck vessels: an experience of consecutive 20 cases without using surgical microscope].

Twenty consecutive cases of pharyngoesophageal cancer who underwent free jejunal reconstruction were reported. The common carotid or external carotid artery was used for a feeder of the free graft. The internal jugular vein were served as a drainage vein. All anastomoses were performed in an end-to-side fashion without using surgical microscopes. Mean carotid artery clamping time was 16 minutes and no neurological complications were noticed postoperatively. Graft failure was occurred in 1 patient. The presenting technique, showing 95% success rate, is recommended as a simple option for vascular anastomosis in free jejunal reconstructive surgery.

Cytokine production in chorioamnionitis.

Lymphohematopoietic cytokines play a significant role in many biological mechanisms including a number of reproductive processes such as ovulation, implantation, placentation, cervical dilation and parturition. Recent experiments have suggested that cytokines play a crucial role in the mechanisms of preterm labor and delivery, which are the leading causes of perinatal morbidity and mortality. Growing evidence suggests that infection is deeply concerned in the pathogenesis of preterm labor and delivery. Chorioamnionitis, a subset of intrauterine infection, has been identified in 20-33% of women with preterm delivery, and the inflammatory and related cytokines, interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8), showed substantial increases in the amniotic fluid at women with intrauterine infection. Although the precise mechanism for chorioamnionitis-driven preterm labor mediated via cytokines is still unknown, both IL-1 and TNF-alpha along with IL-6 enhance prostaglandin production by human amnion cells, chorionic cells and decidual cells. Analysis of the regulatory sequences in the 5' upstream regions of receptor gene for human oxytocin, a potent uterotonic agent, suggests a close relationship between preterm labor and inflammatory cytokines through induction at the oxytocin receptor. Prompt identification of the patients with intra-amniotic infection may be useful in clinical practice. At present, the measurement of IL-8 in maternal serum or the measurement of IL-6 in cervical secretion may be helpful as a non-invasive screening for chorioamnionitis.

cDNA cloning of bradykinin-potentiating peptides-C-type natriuretic peptide precursor, and characterization of the novel peptide Leu3-blomhotin from the venom of Agkistrodon blomhoffi.

A cDNA clone, 1.8 kb long, was isolated from a venom gland cDNA library of Agkistrodon blomhoffi that encodes a large plurifunctional precursor composed of 263 amino-acid residues. Nucleotide sequence analysis of this clone revealed that sequences which code for blomhotin and a novel peptide Leu3-blomhotin are located in the N-terminal region of the precursor polypeptide, followed by four tandemly aligned sequences which code for three types of bradykinin-potentiating peptide. In the C-terminal region, the sequence for the C-type natriuretic peptide was located along with a preceding processing signal. The deduced amino-acid sequences for the four bradykinin-potentiating peptides coincided exactly with previously known sequences for potentiator B, potentiator C and potentiator E. The actual Leu3-blomhotin peptide was subsequently isolated from the venom of A. blomhoffi and characterized. Leu3-blomhotin possesses contractile activity in isolated rat stomach fundus smooth muscle in the same manner as blomhotin. Furthermore, it was shown that blomhotin and Leu3-blomhotin retained activity to inhibit the angiotensin-converting enzyme.

Amino acid sequence studies on cytolytic toxins from sea anemone Heteractis magnifica, Entacmaea quadricolor and Stichodactyla mertensii (Anthozoa).

The complete amino acid sequence of a cytolytic toxin, HmT, isolated from sea anemone Heteractis magnifica was determined. It is composed of 177 amino acid residues and lacks half-cystines. Partial N-terminal sequences of three other cytolysins from Entacmaea quadricolor (EnT) and Stichodactyla mertensii (SmT-1 and SmT-2) were also determined. Comparing these sequences with those of other sea anemone cytolysins, a high degree of homology was observed.

Transient thyrotoxicosis and hypothyroidism following administration of the GnRH agonist leuprolide acetate.

A 45-year-old women with chronic idiopathic thrombocytopenic purpura was given monthly injections of the GnRH agonist leuprolide acetate for the treatment of uterine leiomyoma. Two weeks after the fifth injection, she showed mild symptoms of thyrotoxicosis. At that time, serum thyroxin (T4) and triiodothyronine (T3) levels were elevated whereas TSH level was suppressed. Anti-thyroglobulin (anti-Tg) and anti-thyroid peroxidase (anti-TPO) antibodies were positive, whereas TSH binding inhibitory immunoglobulin (TBII) was undetectable. Two months later, serum T4 and T3 levels spontaneously decreased below the normal ranges. Five months after the onset of the disease, they returned to normal without any treatment. Anti-TPO and anti-Tg antibodies gradually decreased during the clinical course. Thus, the present case was indicated to be an instance wherein silent thyroiditis developed after leuprolide acetate administration. This is the first report to demonstrate the association of thyroid disorder with leuprolide injection.

Satisfaction of patients treated surgically for intractable aspiration.

STUDY OBJECTIVE: Impaired laryngeal protective function can result in intractable aspiration requiring surgical treatment. There are, however, few reports evaluating the satisfaction of patients and the efficacy of surgical therapy. The purpose of this study is to determine whether surgery for intractable aspiration is beneficial for alleviating depression and improving the mood of patients who have undergone surgical treatment and whether patients and their families are satisfied with the therapy. PATIENTS AND STUDY DESIGN: Seven patients with recurrent aspiration pneumonia that could not be controlled by appropriate medical therapies participated in the study. These patients had no hope of recovering laryngeal function. Six underwent laryngectomy and one underwent laryngotracheal separation. After surgery, we evaluated the efficacy of the therapy and the patients' satisfaction with the therapy. METHODS: The following clinical variables concerning surgical procedure were examined: operation time, time until oral intake, videofluorographic study, and surgical complications. The treatment methods including feeding status were also examined before and after surgery. In addition, the following markers were examined to evaluate the efficacy of the surgery: score of aspiration pneumonia, body mass index, total protein, albumin, hematocrit, WBC count, C-reactive protein, erythrocyte sedimentation rate, and the Barthel Index, an indicator of daily activity. Furthermore, the grade of depression and mood, and satisfaction of patients and their caretakers among family members were scored by the Zung self-rating depression scale, a 20-picture face scale, and the visual analog scale. RESULTS: After surgical therapy, we confirmed by videofluorography that aspiration was completely prevented. No surgical complications occurred. By 18 +/- 6 days, all seven patients were able to ingest a meal orally. The need for extensive medical care and repeated hospitalizations became unnecessary after surgery. The control of pneumonia and albumin improved significantly. The grade of depression and mood of patients and their families also improved significantly. Satisfaction scores of patients receiving therapy were very high. CONCLUSIONS: Our study shows that surgical therapy to prevent aspiration improves the depression and mood of patients and their families as well as feeding status and clinical outlook. Surgical therapy for patients with intractable aspiration is effective and beneficial.

Suicide gene therapy for human uterine adenocarcinoma cells using herpes simplex virus thymidine kinase.

In gene therapy, the herpes simplex virus thymidine kinase (HSV-tk) gene is widely used as a suicide agent. Tumor cells expressing HSV-tk are sensitive to nucleoside analogs such as ganciclovir (GCV). An advantage of this system is the bystander killing effect whereby HSV-tk-positive cells exposed to GCV are lethal to surrounding HSV-tk-negative cells. We transfected the HSV-tk gene into a human cervical adenocarcinoma cell line, BU25TK-, and a human endometrial adenocarcinoma cell line, HHUA, by the Lipofectine method. The sensitivity of HSV-tk-positive cells to GCV and bystander killing effect on HSV-tk-negative cells were examined in vitro. HSV-tk-positive cells were sensitive to GCV at concentrations of 1 to 100 microg/ml in a dose- and time-dependent manner. The growth of HSV-tk-negative cells was inhibited when the population of cultured cells contained more than about 3% HSV-tk-positive cells. Moreover, for BU25TK- cells, HSV-tk-positive cells were injected into SCID mice subcutaneously and the effects of GCV therapy and bystander killing at a daily concentration of 25 mg/kg for 14 days were examined. HSV-tk-positive tumors transduced into SCID mice almost disappeared upon GCV treatment. Furthermore, tumor reduction was observed when mixtures of HSV-tk-negative cells containing more than 20% HSV-tk-positive cells were injected into SCID mice. In conclusion, the HSV-tk/GCV system might be applied to both cervical and endometrial adenocarcinoma.

cAMP stimulates the bystander effect in suicide gene therapy of human choriocarcinoma.

In gene therapy, tumor cells expressing herpes simplex virus thymidine kinase (HSV-tk) are sensitive to ganciclovir (GCV) and HSV-tk positive cells exposed to GCV are lethal to adjacent HSV-tk negative cells. This phenomenon has been called the bystander effect, and the gap junction is thought to mediate it. In this study, sensitivity to GCV and bystander effect in a human choriocarcinoma cell line, BeWo, transfected with HSV-tk were investigated. Furthermore, the effect of 8-bromo-cAMP on bystander effect and connexin40 gene transcription were examined. HSV-tk positive cells were sensitive to GCV at the concentration of 10 micrograms/ml in a time-dependent manner. The growth of HSV-tk negative cells was inhibited when the population of cultured cells contained more than 10% HSV-tk positive cells and 8-bromo-cAMP enhanced bystander effect. 8-bromo- cAMP increased connexin40 mRNA expression and gap junctional intercellular communication.

Modulation of normal human eosinophil chemotaxis in vitro by herbimycin A, erbstatin and pervanadate.

BACKGROUND: The mediators involved in eosinophil accumulation in diseases such as allergy continue to be an area of interest, even though little is known regarding the signaling involved in the human cell type recruitment. In the present study, we demonstrate a novel modulatory role of tyrosine kinase and tyrosine phosphatase activities on normal human eosinophil chemotaxis induced by different groups of chemoattractant. METHODS: Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Chemotactic activities against platelet-activating factor (PAF), vasoactive intestinal peptide (VIP) and eotaxin were assessed using a 48-well microchemotaxis chamber assay. Purified eosinophils were pretreated with herbimycin A, erbastatin or pervanadate to examine the role of tyrosine kinase in chemoattractant signaling. RESULTS: Pretreatment of eosinophils with the tyrosine kinase inhibitors herbimycin A and erbstatin significantly blocked chemotaxis induced by eotaxin whilst both inhibitors augmented chemotaxis induced by VIP; however, they had no effect on PAF-induced chemotaxis. On the other hand, pretreatment of eosinophils with the phosphotyrosine phosphatase inhibitor pervanadate resulted in augmentation of eotaxin-induced chemotaxis and inhibition of VIP-induced chemotaxis, but it had no effect on PAF-induced chemotaxis. CONCLUSIONS: These results suggest that protein kinase plays a modulatory role in eosinophil chemotaxis induced by various chemoattractants.

Activated mast cells release extracellular type platelet-activating factor acetylhydrolase that contributes to autocrine inactivation of platelet-activating factor.

IgE-dependent and -independent activation of mouse bone marrow-derived mast cells (BMMC) elicited rapid and transient production of platelet-activating factor (PAF), which reached a maximal level by 2-5 min and was then degraded rapidly, returning to base-line levels by 10-20 min. Inactivation of PAF was preceded by the release of PAF acetylhydrolase (PAF-AH) activity, which reached a plateau by 3-5 min and paralleled the release of beta-hexosaminidase, a marker of mast cell exocytosis. Immunochemical and molecular biological studies revealed that the PAF-AH released from activated mast cells was identical to the plasma-type isoform. In support of the autocrine action of exocytosed PAF-AH, adding exogenous recombinant plasma-type PAF-AH markedly reduced PAF accumulation in activated BMMC. Furthermore, culture of BMMC with a combination of c-kit ligand, interleukin-1beta and interleukin-10 for > 24 h led to an increase in plasma-type PAF-AH expression, accompanied by a reduction in stimulus-initiated PAF production. Collectively, these results suggest that plasma-type PAF-AH released from activated mast cells sequesters proinflammatory PAF produced by these cells, thereby revealing an intriguing anti-inflammatory aspect of mast cells.

Specificity of two types of phospholipase A2 inhibitors from the plasma of venomous snakes.

Specificity of two different types of phospholipase A2 (PLA2) inhibitory proteins from the blood plasma of venomous snakes was investigated. Two Crotalidae inhibitors, having a carbohydrate recognition domain (CRD) in their sequences, inhibited specifically the group-II acidic PLA2s of their own snake venom. On the other hand, Elapidae inhibitor, having two tandem patterns of cysteine residues found in proteins of the Ly-6 superfamily, inhibited not only the group-I PLA2 from its own snake venom but also the group-I, -II, and -III PLA2s from other snake venom. Amino acid sequences of PLA2s that were specifically inhibited by the inhibitors were compared with those of the other PLA2s. A unique aromatic patch structure appeared on the group-II acidic PLA2s was suggested to be involved in the binding to the Crotalidae inhibitors; and residues located in or close to the Ca2+ binding loop of PLA2, in the binding to the Elapidae inhibitor.

Amino acid sequence of two neurotoxins from the venom of the Egyptian black snake (Walterinnesia aegyptia).

The venom of the Egyptian black snake Walterinnesia aegyptia contains at least three toxins, which act postsynaptically to block the neuromuscular transmission of isolated rat phrenic nerve-diaphragm and chicken biventer cervicis muscle. The complete amino acid sequence of the two toxins, W-III and W-IV, consisting of 62 amino acid residues, was elucidated by Edman degradation of fragments obtained after Staphylococcus aureus protease and prolylpeptidase digestion. Although the toxins exhibit close structural homology to other short-chain postsynaptic neurotoxins from Elapidae venoms, toxin IV is unique by having a free SH-group (cysteine) at position 16. In position 35 of W-III, which is located at the tip of the central loop, threonine is replaced by lysine, which may alter the interaction of the toxin with the acetylcholine receptor, since the toxin is seven times less lethal than toxin W-IV.

Lactoferrin as a suppressor of cell migration of gastrointestinal cell lines.

The effects of lactoferrin (Lf), an iron-binding glycoprotein, on cell migration were investigated. Lf inhibited the cell migration of three gastrointestinal cell lines (Caco-2 cells, AGS cells, and IEC-18 cells) in vitro. Both iron-saturated (holo) and iron-depleted (apo) Lf showed this inhibitory effect. Chelation of iron in the culture medium by desferrioxamine did not affect the activity of either form of Lf. A pepsin hydrolysate of Lf exhibited effectiveness similar to that of intact Lf. These results demonstrate a novel activity of Lf and suggest a potential role for this molecule in gastrointestinal wound healing, which is independent of its iron-binding capacity.

The novel role of 3',5'-guanosine monophosphate (cGMP) on the differentiation of trophoblasts: comparison with the effects of 3',5'-adenosine monophosphate (cAMP).

We investigated the effects of 3',5'-guanosine monophosphate (cGMP) on the differentiation of human trophoblasts. Isolated cytotrophoblasts were cultured with 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) or 8-bromoadenosine 3'5-cyclic monophosphate (8-Br-cAMP) and then stained immunocytochemically with anti-human chorionic gonadotropin (anti-hCG) antibody to identify hCG expression as an index of differentiation. Concurrently, morphological changes from cytotrophoblasts to syncytiotrophoblasts were analyzed. Both 8-Br-cGMP and 8-Br-cAMP enhanced the expression of hCG in cultured cytotrophoblasts with the differentiation of cytotrophoblasts to syncytiotrophoblasts dose-dependently. With regard to trophoblast proliferation, 8-Br-cAMP but not 8-Br-cGMP enhanced [3H]thymidine uptake by these cells. hCG, a trophoblast-specific glycoprotein hormone has been identified as a potent growth factor for trophoblasts, also increased [3H]thymidine uptake and the intracellular 3',5'-adenosine monophosphate (cAMP) concentration. However, in this study, hCG did not increase the concentration of intracellular cGMP. We also showed that sodium nitroprusside (SNP), which is a donor of nitric oxide (NO), enhanced intracellular cGMP concentration. These results suggest that cGMP enhances trophoblast differentiation without affecting their proliferation, while cAMP enhances both differentiation and proliferation. We conclude that an alternative pathway mediated through cGMP is responsible for the differentiation of trophoblasts. NO may be involved in trophoblast differentiation with an increase in cellular cGMP level.

Inhibition of lysoPAF acetyltransferase activity by flavonoids.

OBJECTIVE AND DESIGN: Several kinds of flavonoids, widely distributed natural products of the vegetable kingdom which possess anti-inflammatory activity, were examined for inhibitory effects on the acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF) acetyltransferase activity. METHODS: Acetyl-CoA:lysoPAF acetyltransferase activity was determined using homogenates of a rat mucosal-type mastocytoma cell line, RBL-2H3 as an enzyme source. The production of platelet-activating factor (PAF) in rat peripheral white blood cells stimulated with the calcium ionophore A23187 was studied. RESULTS: Of the flavonoids tested, luteolin and quercetin exhibited significant inhibitory effects (IC50, 45 microM and 80 microM, respectively), whereas other structurally-related flavonoids failed to affect the lysoPAF acetyltransferase activity. Luteolin did not suppress the activity of choline acetyltransferase, suggesting that the inhibition observed here was specific. Luteolin also inhibited the production of PAF in rat peripheral white blood cells. CONCLUSIONS: These results indicate that luteolin could become a leading compound for developing a novel type of anti-inflammatory, anti-allergic drugs that target lysoPAF acetyltransferase.

Local production of interleukin-5 by T lymphocytes is associated with recruitment of eosinophils in patients with eosinophilic granuloma of the soft tissue.

Eosinophilic granuloma of the soft tissue (EOSG) is a rare disease of unknown cause. Since the in vivo mechanism of eosinophilia remains unclear, the present study was performed to investigate the mechanism of the infiltration of eosinophils into the granuloma tissue. Immunohistochemical techniques and an eosinophil chemotactic assay were used in analysis. Peripheral blood eosinophils obtained from one patient showed an increased chemotactic response against tissue extract that was inhibited by pretreatment with anti-IL-5 antibodies. Eosinophils obtained from the peripheral blood of patients with EOSG showed a significant increase in chemotactic activity toward 10(-9) M recombinant human (rh) IL-5 versus that of healthy individuals, whereas eosinophils from granuloma tissue showed no chemotactic response toward rhIL-5, indicating that IL-5 may deactivate the eosinophils. Immunohistochemical studies showed that CD4+ cells were predominantly found in the extrafollicular region, along with interleukin-5+ (IL-5) cells. Staining of the adjacent 3-micrometers sections for CD3, eosinophils, and IL-5 revealed that most of the IL-5 immunoreactive CD3+ cells exhibited cytoplasmic staining. Conversely, 97% of IL-5+ eosinophils were stained peripherally in a ring-like manner, suggesting that IL-5 was bound to its cell surface receptor on the eosinophil. IL-5 mRNA expression was detectable in the CD3+T lymphocytes but not in eosinophils from granuloma tissue. These findings suggest that locally produced IL-5 from T lymphocytes may enhance the infiltration of eosinophils into the eosinophilic granuloma.

Kappa-casein suppresses melanogenesis in cultured pigment cells.

The effects of bovine milk proteins on melanogenesis in B16 cells were examined. Both whey protein isolate and casein exhibited depigmenting properties. Among the major protein components of milk--including beta-lactoglobulin, alpha-lactalbumin, alpha-, beta-, and kappa-casein--only kappa-casein exhibited the depigmenting effect. However, the carboxyl terminal peptide of kappa-casein, glycomacropeptide, did not show this activity. Also, kappa-casein promoted the proliferation of the cells and inhibited the activity of tyrosinase in the cells. These results indicate that kappa-casein acts as a melanogenesis-suppressing modulator.

Leukemia inhibitory factor (LIF) enhances trophoblast differentiation mediated by human chorionic gonadotropin (hCG).

We investigated the effect of LIF on the differentiation of trophoblasts. Isolated cytotrophoblasts were cultured with and without LIF and cell smears were immunocytochemically analyzed, using anti-hCG antibody. The percentage of differentiated trophoblasts stimulated by 10ng/ml of LIF was about 2.5-fold that in the control culture. The effect of LIF in inducing the differentiation of cytotrophoblasts to syncytiotrophoblasts was dose-dependent. The same effect was shown when hCG was added to the medium. This enhancing effect of LIF on trophoblast differentiation was blocked by adding anti-hCG antibody to the culture system. These results indicate that LIF enhanced trophoblast differentiation by stimulating hCG production in trophoblasts, and not by exerting a direct effect on the trophoblasts.