Effects of bisphosphonates on human esophageal squamous cell carcinoma cell survival.

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers in Japan. Anticancer chemotherapy has been useful for ESCC treatment. However, therapeutic options are limited. Recently, bisphosphonates (BPs), which are osteoporosis drugs, have shown anticancer effects in several cancer cell lines, but the effects against ESCC cell lines are unknown. In this study, we examined the cytotoxic effects of BPs and their mechanisms of cytotoxicity in human ESCC cell lines. A first-generation BP (etidronate), two second-generation BPs (alendronate and pamidronate), and two third-generation BPs (risedronate and zoledronate) were used in this study. All BPs, except etidronate, were cytotoxic, as indicated by increased caspase-3/7 activity and numbers of Annexin-fluorescein isothiocyanate positive cells in ESCC cell lines. From cell cycle analysis, G0/G1-phase arrest was observed upon treatment with second- and third-generation BPs. In addition, Cyclin D1 protein expression levels were decreased by second- and third-generation BP treatment. Although squalene and trans, trans-farnesol minimally affected BP cytotoxicity, treatment with geranylgeraniol inhibited BP cytotoxicity almost completely. We concluded that second- and third-generation BPs are cytotoxic to ESCC cell lines as they induce apoptosis and inhibit the cell cycle through mevalonate pathway inhibition. Therefore, BP treatment may be a beneficial therapy in ESCC patients.

Cyclosporin A treatment and decreased fungal load/antigenemia in experimental murine paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb). The cyclosporin A (CsA) is an immunosuppressant drug that inhibits calcineurin and has been described as a potential antifungal drug. The present study investigated the effect of CsA on the immune response, fungal load/antigenemia in experimental murine PCM. It was used four groups of BALB/c mice: (a) infected with 1 x 105 Pb18 yeast cells (Pb), (b) infected and treated with CsA every other day 10 mg/kg of CsA (s.c.) during 30 days (Pb/CsA), (c) treated with CsA (CsA) and (d) no infected/treated (PBS). The immune response was evaluated by lymphocyte proliferation, DTH assays to exoAgs, ELISA for IgG anti-gp43 (specific immune responses) and cytokine serum levels (IFN-γ, TNF-α, IL-4 and IL-10). Fungal load was determined by lung colony-forming units (CFU) counts, lung and liver histopathology analysis and antigenemia determined by inhibition-ELISA. As expected, CsA was able to inhibit the specific cellular and humoral immune response (P < 0.05), with decrease in serum IFN-γ, TNF-α and IL-4 levels (P < 0.05). Cyclosporin A treatment also resulted in significantly decreased lung Pb CFU (P < 0.05) as well as a lower number of yeasts in the lung and liver (P < 0.05) by histopathology. In concordance, the decreased antigenemia was observed in Pb/CsA group (P < 0.05). In conclusion, even with immunosuppressive action, treatment with CsA results in decreased lung fungal load/antigenemia in experimental PCM in BALB/c mice. Further study is required to determine whether this represents less severe disease or protection by CsA.

Increased susceptibility to cardiovascular effects of dihydrocapcaicin in resuscitated rats. Cardiovascular effects of dihydrocapsaicin.

BACKGROUND: Survivors of a cardiac arrest often have persistent cardiovascular derangements following cardiopulmonary resuscitation including decreased cardiac output, arrhythmias and morphological myocardial damage. These cardiovascular derangements may lead to an increased susceptibility towards the external and internal environment of the cardiovascular system as compared to the healthy situation. METHODS: Here we tested the hypothesis that the cardiovascular system in healthy rats and rats resuscitated from a cardiac arrest may be differentially affected by a transient receptor potential vanilloid type 1 agonist, by continuous intravenous infusion of dihydrocapsaicin (DHC). RESULTS: Compared to baseline, infusion of DHC caused an initial increase in mean arterial blood pressure in both healthy and resuscitated rats of 25% and 10%, respectively. Also, we observed an initial response of tachycardia in both healthy and resuscitated rats of 30% and 20%, respectively. Then, at high levels of DHC infusion (> 2.0 mg/kg/hr) we observed two single episodes of transient bradycardia and hypotension in 33% of the healthy rats, which was consistent with a TRPV1 agonist induced Bezold-Jarisch reflex. In contrast, in resuscitated rats we observed multiple episodes of bradycardia/hypotension in 100% of the rats and at a dose of DHC of 0.65 mg/kg/hr. Notably, this DHC effect could be completely blocked in the resuscitated rats by pre-treatment with atropine, a muscarinic acetylcholine antagonist. CONCLUSIONS: Our results indicate that the susceptibility of the rats towards TRPV1 agonist induced Bezold-Jarisch reflex is increased in those resuscitated from cardiac arrest compared to the healthy situation.

Drug-induced mild therapeutic hypothermia obtained by administration of a transient receptor potential vanilloid type 1 agonist.

BACKGROUND: The use of mechanical/physical devices for applying mild therapeutic hypothermia is the only proven neuroprotective treatment for survivors of out of hospital cardiac arrest. However, this type of therapy is cumbersome and associated with several side-effects. We investigated the feasibility of using a transient receptor potential vanilloid type 1 (TRPV1) agonist for obtaining drug-induced sustainable mild hypothermia. METHODS: First, we screened a heterogeneous group of TRPV1 agonists and secondly we tested the hypothermic properties of a selected candidate by dose-response studies. Finally we tested the hypothermic properties in a large animal. The screening was in conscious rats, the dose-response experiments in conscious rats and in cynomologus monkeys, and the finally we tested the hypothermic properties in conscious young cattle (calves with a body weight as an adult human). The investigated TRPV1 agonists were administered by continuous intravenous infusion. RESULTS: Screening: Dihydrocapsaicin (DHC), a component of chili pepper, displayed a desirable hypothermic profile with regards to the duration, depth and control in conscious rats. Dose-response experiments: In both rats and cynomologus monkeys DHC caused a dose-dependent and immediate decrease in body temperature. Thus in rats, infusion of DHC at doses of 0.125, 0.25, 0.50, and 0.75 mg/kg/h caused a maximal ΔT (°C) as compared to vehicle control of -0.9, -1.5, -2.0, and -4.2 within approximately 1 hour until the 6 hour infusion was stopped. Finally, in calves the intravenous infusion of DHC was able to maintain mild hypothermia with ΔT > -3°C for more than 12 hours. CONCLUSIONS: Our data support the hypothesis that infusion of dihydrocapsaicin is a candidate for testing as a primary or adjunct method of inducing and maintaining therapeutic hypothermia.

Detection of Paracoccidioides brasiliensis gp43 gene in sputa by loop-mediated isothermal amplification method.

The fungus Paracoccidioides brasiliensis is the pathogen of paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. The loop-mediated isothermal amplification method (LAMP) was used in this study to detect the presence of P. brasiliensis in sputa samples from patients with chronic PCM, suspected PCM, and a negative control. The target P. brasiliensis gp43 gene was amplified in less than 4 hr in 11 of 18 sputa samples tested. The LAMP method had the advantage of speed and simplicity compared with the classic diagnostic methods such as the histopathological test or biological material culture and did not require sophisticated technical apparatus. It would be an important aid in cases where immediate treatment would mean patient survival, especially in immune-suppressed patients.

Comparison of the stimulating capacity of human leukemia T-cell, null cell, and B-cell lines in mixed lymphoma culture.

One-way mixed lymphocyte reactions between lymphocytes from normal human donors and mitomycin C-treated cells from human leukemia T-cell, null cell, and B-cell lines were investigated. An Epstein-Barr virus (EBV)-negative Japanese Burkitt's lymphoma line and two EBV-positive normal lymphoid cell lines were studied in parallei. Normal lymphocytes were stimulated significantly by the cultured null cells and B-cells, but only slightly by the cultured T-cells. The stimulatory capacity of these two leukemia cell lines was approximately equal to that of the lymphoma and normal B-cell lines. The results suggest that not only leukemia B-cells but also leukemia null cells have stimulatory determinants in mixed lymphocyte culture.

Meningioma with hypergammaglobulinemia. Case report.

The case of a 14-year-old boy presenting with hypergammaglobulinemia (immunoglobulin (Ig)G and IgA) and a cerebellopontine angle tumor is reported. The tumor was histologically confirmed as meningioma infiltrated with plasma cells, lymphocytes, and histiocytes. Immunohistochemical studies showed that the infiltrating plasma cells were stained by antibodies to IgG and IgA. After total tumor removal, the hypergammaglobulinemia immediately resolved. A plausible interpretation of this sequence of events is that the inflammatory cell reaction to the meningioma caused the immunological response followed by an unusual hypergammaglobulinemia.

Isolation of neocopiamycin A from Streptomyces hygroscopicus var. crystallogenes, the copiamycin source.

Fermentation of Streptomyces hygroscopicus var. crystallogenes, the copiamycin source, yielded several minor components with antifungal activity. One of these minor components, neocopiamycin A, was isolated and characterized. The structure of neocopiamycin A was determined as N-demethylcopiamycin on the basis of spectroscopic evidence. The antibiotic was found to be more active against Gram-positive bacteria and fungi but less toxic than copiamycin.

Effects of streptonigrin derivatives and sakyomicin A on the respiration of isolated rat liver mitochondria.

Though all the streptonigrin derivatives with modifications in the carboxyl group on C2' were active as electron acceptors in the oxidation of NADH by Clostridium kluyveri diaphorase as well as streptonigrin, the glycine derivative did not show any marked effect on the KCN-insensitive oxidation of glutamate by rat liver mitochondria, suggesting a poor membrane transport of the glycine derivative. Sakyomicin A also induced the KCN-insensitive oxidation of glutamate by mitochondria, while a trace activity was observed by mitomycin C and the effect of doxorubicin was negligible. Like streptonigrin, the autoxidation of a reduced form (hydroquinone) of sakyomicin A to a quinone accompanied the generation of H2O2. However, exogenous NADH was oxidized by mitochondria in the presence of sakyomicin A but not streptonigrin.

Heterotransplantation of human leukemic B-cell, T-cell and null-cell lines in hamsters.

Human leukemic B-cell (BALL-1), T-cell (TALL-1) and null-cell (NALL-1) lines have been established from three patients with acute lymphoblastic leukemia (ALL). To study the heterotransplantability and in vivo growth characteristics, attempts were made to transplant these ALL cell lines into newborn Syrian hamsters treated with rabbit anti-hamster thymocyte serum. Intraperitoneal implantation of 1.8-3.5 x 10(7) cells gave rise to invasive tumors in all recipients after 15 to 41 days. In addition to a common in vivo feature of mesenteric and retroperitoneal tumors, BALL-1 line was characterized by infiltration of the skin, massive ascites and bone marrow invasion. TALL-1 cells infiltrated various organs including the lymph nodes, liver, gallbladder, spleen, bone marrow, central nervous system and eyes. NALL-1 line grew slowly, producing the least tumors, although there were distant metastases in the lungs. Tumor cells were detected in the blood of 2 of 3 BALL-1-bearing hamsters and in the blood of 4 of 5 TALL-1-bearing hamsters. Thus, these three ALL cell lines were found to exhibit a characteristic biological behavior in hamsters, which might be related to the different cell lineage.

Isolation of Candida dubliniensis from the oral cavity of an HIV-positive child in Brazil.

Candida dubliniensis is a newly-recognized Candida species and an important infectious pathogen, particularly for HIV-positive patients. >From oral smear samples from the radix linguae of 173 HIV-positive children, we obtained four yeast isolates which took a blue-green color on CHROMagar Candida plate at 37 degrees C for 48 hours from one HIV-positive 3-year-old boy in Brazil. The isolates were difficult to grow on potato dextrose agar plate at 42 degrees C, produced abundant chlamydospores on a cornmeal agar plate with Tween 80, and sprouted germ tubes in saline with horse serum, and the antigenic profile by CANDIDA CHECK test was useless. Carbohydrate assimilation tests by ID32C showed no reference code number in the reference book. The isolates were subjected to molecular biological assay of the DNA sequence of the large-subunit ribosomal DNA region (D1/D2) and randomly amplified polymorphic DNA (RAPD). The DNA sequence agreed with those of standard C. dubliniensis strains, and therefore, the isolates were identified as C. dubliniensis. RAPD band pattern analysis indicated that the clinical isolates might summarize one genotype. Although the child did not present oral lesions, the fungus might be latent for opportunistic infection.

Atypical Cryptococcus neoformans isolate from an HIV-infected patient in Brazil.

Cryptococcus neoformans is an important fungal pathogen in immunocompromised hosts. Capsulation, urease and melanin synthesis activity of the fungus are well known virulence factors. Although artificial melanin-deficient mutants of Cr. neoformans have been investigated, the clinical mutant is rare. We found a Cr. neoformans isolate in the cerebrospinal fluid of an AIDS patient which produced a light tan colony on a caffeic acid cornmeal agar (CACA) plate. The mycological feature of the isolate was as follows; normal capsulation, defective inositol assimilation ability, serotype A; urease-positive; mating type alfa; haploid; extremely slow growth in RPMI 1640 medium, Sabouraud dextrose broth, brain heart infusion broth and yeast nitrogen base; lower production of melanin with L-DOPA substrate; and low virulence to ddY mice. We also investigated the partial DNA sequence of CNLAC1 gene between the 3085th to 3623rd base. There were many substitutions, 3 insertions and 3 deletions in the isolate compared with GenBank accession number L22866. The result indicated some functional disorder in the gene. Although the CACA plate is an excellent selective medium for Cr. neoformans, other identification methods should also be used.

[The relation of retinal vascular occlusion and carotid artery stenosis].

We evaluated carotid ulceration and stenosis in 16 patients with central retinal artery occlusion (CRAO), 13 patients with branch retinal artery occlusion (BRAO), and 27 patients with central retinal vein occlusion (CRVO). One of three kinds of angiography was selected according to the patient's condition. These included conventional cerebral angiography, intravenous digital subtraction angiography (IVDSA), and magnetic resonance angiography (MRA). Carotid artery stenosis (50% or greater) was noted in 37.5% (6/16) of the patients with CRAO and in 23.1% (3/13) of the patients with BRAO. Only one of 27 patients with CRVO had a contralateral 50% carotid artery stenosis. In order to detect an underlying systemic disease in evaluating patients with retinal artery occlusion, examination of the carotid vessels is essential. It may also be helpful to estimate the risk of cerebral stroke for patients with retinal stroke.

[In vitro susceptibility of pathogenic Nocardia to beta-lactam antibiotics, especially imipenem, a carbapenem antibiotic].

In vitro antibacterial activity of 30 beta-lactam antibiotics including 2 beta-lactamase inhibitors (clavulanic acid and sulbactam) against 2 major pathogenic Nocardia, i.e. Nocardia asteroides group and Nocardia brasiliensis was studied. Among the antibiotics tested, a newly developed carbapenem antibiotic, imipenem (IPM), was found to be the most active, followed by oxacephem group antibiotic flomoxef (FMOX). IPM exhibited activity against only N. asteroides group (N. asteroides, Nocardia farcinica and Nocardia nova). On the other hand, FMOX showed activity against all pathogenic Nocardia tested. A 2- to 30-fold decreases in minimum inhibitory concentration (MIC) for N. asteroides, N. brasiliensis and N. farcinica was noted when antibiotics and beta-lactamase inhibitors were combined compared to antibiotics alone. Further combination and enzymatic studies indicated that all pathogenic Nocardia possess beta-lactamase except for a half of the strains of N. nova. These species' specific sensitivity patterns of pathogenic Nocardia are discussed in this paper with references to their taxonomic positions.

[C-P angle epidermoid carcinoma: a case report].

The patient, a 39-year-old man, underwent initial surgery for total removal of a left C-P angle tumor histologically diagnosed as epidermoid carcinoma. Postoperative irradiation therapy was administered over the whole brain and spinal cord, 50 Gy and 20 Gy respectively. The patient was then released without symptoms. 15 months later, he was readmitted for paraparesis and urinary retention. CT scan revealed no tumor recurrence in the intracranial area but did show intraspinal cyst of the lower thoracic level. After cyst-subarachnoid shunt, severe acute hydrocephalus was shown on CT scan. His general condition progressively deteriorated. Ten days after the shunt operation, he died of systemic bleeding tendency and cachexia. During surgery, the authors obtained dark yellowish fluid from the intraspinal cyst. Gene survey of the specimen from the cyst wall disclosed abnormality. It is important in the diagnosis of primary intracranial epidermoid carcinoma that we rule out the existence of extracranial cancer and direct invasion through the dura. Though epidermoid carcinoma can be fatal, radiotherapy was an efficacious treatment in the present case, as well as in three other reported cases.

[Experimental study of brain stem infarction in dogs--effect on BAEP, SSEP, blink reflex and EEG of perforator occlusion].

Assessment of the lesion in the brain stem by evoked potentials has not been well established. We have already developed a model of brain stem ischemia by occluding the perforators of the posterior cerebral arteries of the dog. The ischemic lesions locates mainly in the ventral side of the midbrain. Using this model, we assessed brain stem function by brain stem auditory evoked potential (BAEP), surface- and depth-recorded (in medial lemniscus) short latency somatosensory evoked potential (SSEP), blink reflex (BR) and electroencephalography (EEG), and investigated the correlation between the electrophysiological abnormalities and the lesion in the brain stem. The studies were performed for 6 hours after perforator occlusion. Furthermore, depth-recorded SSEP and regional cerebral blood flow (rCBF) were measured under induced hypotension by withdrawal of arterial blood. BAEP did not change in 13 of 16 animals. Surface-recorded SSEP remained unchanged in all 6 animals. The results are probably due to the fact that the lesion does not involve the auditory and somatosensory pathways and the accompanying events such as edema does not affect the both pathways. Depth-recorded SSEP remained unchanged after occlusion and did not disappear even when rCBF fell below 10 ml/100 g/min. It may be suggested that the threshold for electrical failure in the brain stem is much lower than that in the cortex. In BR, R1 did not change but ipsilateral R2 became nearly invisible immediately after perforator occlusion in all animals. The fact that the ischemic lesion did not involve the pons and disturbed reticular formation in the midbrain may probably account for the remaining of R1 and the disappearance of ipsilateral R2.(ABSTRACT TRUNCATED AT 250 WORDS)