Colorectal cancer susceptibility variants alter risk of breast cancer in a Chinese Han population.

Recent genome wide association studies (GWAS) and candidate gene studies have revealed many novel loci associated with colorectal cancer susceptibility. We evaluated the effect of these colorectal cancer-associated variants on the risk of breast cancer in a Chinese Han population. Seven single nucleotide polymorphisms (SNPs) (rs3856806 in PPARG, rs7014346 in POU5F1P1, rs989902 in PTPN13, rs1801278 in IRS1, rs7003146 in TCF7L2, rs1503185 in PTPRJ, and rs63750447 in MLH1) were genotyped in Han Chinese subjects, including 216 patients with breast cancer and 216 matched controls, using the Sequenom MassARRAY platform. The association of genotypes with susceptibility to breast cancer was analyzed using the odds ratio (OR), with 95% confidence interval (CI) and logistic regression. Three SNPs (rs7014346, rs989902, and rs7003146) were found to be significantly associated with the susceptibility of breast cancer. The GA and AA genotypes of rs7003146 in TCF7L2, and the CA and CC genotype of rs989902 in PTPN13 were associated with reduced breast cancer risk in the Chinese Han population based on the best-fit dominant model. The GG genotype of rs7014346 in POU5F1P1 was also significantly associated with decreased breast cancer risk under the best-fit additive model. Our results confirmed the association of rs7014346 in POU5F1P1, rs989902 in PTPN13, and rs7003146 in TCF7L2 with variations in the risk of breast cancer in a Chinese Han population.

The use of albendazole and diammonium glycyrrhizinate in the treatment of eosinophilic meningitis in mice infected with Angiostrongylus cantonensis.

Angiostrongylus cantonensis (A. cantonensis) infection causes eosinophilic meningitis in humans. Eosinophilia and a Th2-type immune response are the crucial immune mechanisms for eosinophilic meningitis. CD4+CD25+ regulatory T cells (Treg) are involved in the pathogenesis of A. cantonensis. Diammonium glycyrrhizinate (DG) is a compound related to glycyrrhizin (GL), a triterpene glycoside extracted from liquorice root. We investigated the curative effects and probable mechanisms of therapy involving a combination of albendazole and DG in BALB/c mice infected with A. cantonensis, and compared these with therapy involving albendazole and dexamethasone. We analysed survival time, body weight, signs, eosinophil numbers, immunoglobulin E (IgE), interleukin-5 (IL-5), and eotaxin concentrations, numbers and Foxp3 expression of CD4+CD25+ Treg, worm recovery and histopathology. The present results demonstrated that the combination of albendazole and DG could increase survival time more efficiently and relieve neurological dysfunction; decrease weight loss, eosinophil numbers, concentrations of IgE, IL-5 and eotaxin, the number and expression of Foxp3 of CD4+CD25+ Treg; and improve worm recovery and histopathology changes in treated animals, compared with the combination of albendazole and dexamethasone. The observations presented here suggest that the albendazole and dexamethasone combination could be replaced by the combination of albendazole and DG.

Pharmacokinetics, tissue distribution, and metabolism of novel DNA topoisomerase I inhibitor yuanhuacine in rabbit.

A liquid chromatography/tandem mass spectrometry method was developed and validated for the quantitative determination of yuanhuacine (YHC), a daphne diterpene ortho-ester anticancer agent, and identification of its metabolites. Pharmacokinetic behaviour, tissue distribution, and metabolism were investigated in rabbit. YHC plasma data best fitted to a two-compartment model and were characterized by an elimination half-life t(1/2)(beta) of 11.1 h following intravenous administration. Tissue distribution studies did not identify any tissues having a high affinity for YHC. The main metabolites are proposed to be M392I, M392II, and M390, resulting from the ortho-ester group and aromatic ester bond being cleaved off simultaneously during Phase I metabolism. This investigation contributes to an understanding of the metabolism of daphne diterpene ortho-esters.

[Expression and immunocompetence characterization of Plasmodium falciparum lactate dehydrogenase].

OBJECTIVE: To express lactate dehydrogenase (LDH) gene of Plasmodium falciparum FCC1/HN in the E. coli TG1 and analyse its immunocompetence. METHODS: The LDH gene of the P. falciparum was specifically amplified by polymerase chain reaction, and the recovered gene fragment was cloned into pGEX-4T-1 vector for expression of fusion protein with glutathione S-transferase(GST). The recombinant plasmid was transformed into the E. coli TG1. Four mice (Kunming strain) were immunized with purified expressed protein(antigen) and the polyclonal antibodies were collected. The immunocompetence of recombinant protein was analysed by ELISA and Western-blot. RESULTS: The LDH gene of P. falciparum was successfully expressed in the E. coli TG1. The expressed protein exhibited a specific reaction with immune sera obtained from rabbits immunized with P. falciparum. The specific humoral responses were induced in mice and the titer of the specific antibody was 1:16 by two-dimensional diffusion assay. CONCLUSION: The LDH gene of P. falciparum has been successfully expressed in the E. coli TG1 and the expressed protein has high antigencity.

[Preparation and characterization of McAbs against lactate dehydrogenase of Plasmodium falciparum].

OBJECTIVE: To prepare and characterize the monoclonal antibodies(McAbs) against lactate dehydrogenase of the Plasmodium falciparum(LDHp). METHODS: BALB/c mice were immunized with purified recombinant LDHp and McAbs against LDHp were prepared according to the protocol of hybridoma technique. The McAbs were characterized by ELISA and Western blot analysis. RESULTS: Two McAbs against LDHp antigen were obtained. Both McAbs were IgG2b. The titer of two McAbs(2A5, 1H10) in the ascites was 1:25,600 and 1:12,800, and in supernatant were 1:512,1:256 respectively. The result of ELISA indicated that two McAbs reacted only with P. falciparum, and did not react with normal human red blood cells, P. vivax, Toxoplasma gondii, Schistosoma japonicum. It is recognized 33 kDa protein which was defined as LDHp by Western blot analysis. CONCLUSION: Two hybridoma cell lines secreting high titer of McAbs against LDHp with high specificity were established.

Interferon modulates neuronal activity recorded from the hypothalamus, thalamus, hippocampus, amygdala and the somatosensory cortex.

Neuromodulators interact with classically defined neurotransmitters to regulate a variety of biological processes. The aim of the present study was to study whether interferon-alpha (IFN) can be considered as a neuromodulator. Single cell recordings from five CNS structures were recorded before and following three different routes of IFN administration in Sprague-Dawley rats to substantiate that IFN is a neuromodulator. IFN modulated the majority of the hypothalamic (70%), amygdala (76%), hippocampus (75%) and cortical (82%) cells whether the route of administration was within the brain or given peripherally (i.v. or i.p.). The main difference among the three routes of IFN administration on the neuronal activity of these four CNS sites was the onset of the effect. However, the thalamic neurons responded differently. IFN injection within the brain modulated activity of 43% of thalamic neurons, but only 25% and 17% of the neurons when IFN was given i.v. or i.p., respectively. IFN, in general suppressed hypothalamic neuronal activity while accelerating neuronal activity in all the other studied CNS sites. In conclusion, IFN is an endogenous peptide synthesized and released both peripherally and centrally, with the same effects on neuronal activity whether it is given systemically or locally within the brain. This suggests that IFN can be considered as a neuromodulator.

Lateral hypothalamus: site involved in pain modulation.

The present study is an attempt to examine the neuronal circuitry of a supraspinal site engaged in pain modulation. Five physiological measures were postulated as the criteria for defining a central nervous system site engaged in the circuitry of pain modulation. The lateral hypothalamus met these five measures: (i) 81% of the lateral hypothalamus neurons (247/304) responded to noxious stimuli using a single cell recording procedure; (ii) stimulation of the periaqueductal gray-dorsal raphe area or the habenula modulated 98% and 87% of the lateral hypothalamus noxious-evoked activity; (iii) microiontophoretically applied morphine modulated 77% of the lateral hypothalamus noxious evoked activity; (iv) electrical stimulation of the lateral hypothalamus produced behavioral analgesia proportional to the stimulus intensity as assessed by the tail flick assay; and (v) morphine application into the lateral hypothalamus produced behavioral analgesia in a dose-response manner using the tail flick assay. In conclusion, the lateral hypothalamus can be considered one of the pain modulation sites.

Influence of fetal hippocampal grafts on lesions induced by intraventricular infusion of N-methyl-D-aspartate (NMDA) into rats.

Effects of fetal hippocampal transplants were evaluated following a prolonged intraventricular excitotoxic lesion (1.0 mg of N-methyl-D-aspartate over two weeks infusion) in F344 rats. The septum and ipsilateral hippocampus (CA1 and dentate regions) showed extensive cell loss, decreased acquisition of spatial memory was observed and a decrease in AChE positive fiber innervation to the hippocampus was noted following the lesion. Fetal hippocampal transplants into the posterior lateral ventricle resulted in moderate graft survival and physiological analysis of graft-host interconnection in vitro demonstrated evoked field potentials. However, the transplants did not lead to significant improvement in behavior, possibly due to poor synaptic integration of the intraventricular transplants into the host hippocampus. The prolonged intraventricular NMDA lesion may be helpful to understand a mixed lesion model of both septal areas and hippocampus and also as a background lesion in which to assess the connectivity and development of various types of neural grafts.

Hypothalamic, dorsal raphe and external electrical stimulation modulate noxious evoked responses of habenula neurons.

Extracellular recording techniques were used to investigate the effects of focal brain stimulation and external electrical stimulation on spontaneous activity and on noxious evoked responses in the habenular nucleus of anesthetized Sprague-Dawley rats. Two hundred and forty-one habenular neurons were tested to noxious and non-noxious stimuli. The habenular neurons exhibited three cell types according to their patterns of response to the noxious stimulus: 123 neurons (51%) responded to noxious stimulus by excitation and were classified as "nociceptive-on" cells; 56 neurons (23%) responded to the same noxious stimulus by decreasing their firing rate and were classified as "nociceptive-off" cells; and 62 neurons (26%) failed to respond to noxious stimulation and were classified as "non-nociceptive" cells. None of these 241 cells responded to non-noxious stimulus. One hundred and fifty-five, 160, 142 and 241 habenular neurons were tested following focal lateral hypothalamus stimulation, dorsal raphe stimulation, cerebellar stimulation and transcranial electrical stimulation alone and concomitant with noxious stimulation, respectively. The observations demonstrate that focal lateral hypothalamic, dorsal raphe and external (transcranial) electrical stimulation suppresses habenular noxious evoked responses while cerebellar electrical stimulation elicits no effect on the nociceptive-off cells and augmenting effects on the nociceptive-on cells. In addition, it was observed that low current (below threshold) external transcranial electrical stimulation was as effective in suppression of habenular noxious evoked responses as was focal brain electrical stimulation in the lateral hypothalamus and dorsal raphe.

Focal dorsal raphe stimulation and pinnal electrical stimulation modulate spontaneous and noxious evoked responses in thalamic neurons.

This study investigated the nocieceptive responses of single neurons within the nucleus parafascicularis (PF) thalami of the rat following two modes of electrical stimulation known to induce analgesia. It was found that both focal electrical dorsal raphe stimulation (DRS) and bilateral pinnal (ear) electrical stimulation (PES) converge on the same PF neurons, affecting both the spontaneous discharges and the noxious evoked responses toward these neurons. The effects of different stimulus current intensity, frequency and pulse duration were also examined. It was found that for both DRS and PES at pulse frequency of 10 Hz and current amplitude of 10 microA are the optimal parameters to modulate both the spontaneous and the noxious evoked responses. These stimuli produced prolonged effects related to the duration of stimulation. The external (PES) low current stimulation which was delivered below the sensory threshold was as effective in modulating noxious responses as the invasive DRS in intact animals and in animals with bilateral dorsolateral-funiculus ablation. It was observed that dorsal lateral funiculus ablation (DLFx) did not modify the DRS and the PES effects. These observations further support the existence of an ascending pain modulation pathway.

Trans-cranial electrical stimulation attenuates abrupt morphine withdrawal in rats assayed by remote computerized quantification of multiple motor behavior indices.

The goal of the present study was to assess the effects of trans-cranial electrical stimulation on the behavioral signs of the abrupt withdrawal syndrome of rats. However, this goal also necessitated the introduction of an experimental model measuring animal behavior for prolonged periods of time using a computerized animal activity monitoring system to quantify spontaneous motor activities associated with abstinence behavior. Comparable withdrawal severity was obtained by both the activity monitoring system and investigator observation of motor signs of abstinence behavior. Moreover, using this system we demonstrate a time-dependent effect of electrical stimulation in reducing the severity of various indices of motor hyperactivity associated with abrupt morphine withdrawal in rats.

The rat hippocampal slice preparation as an in vitro model of ischemia.

In vivo models of cerebral ischemia do not fully control for the interacting effects of many variables (e.g., anesthesia, temperature, cerebrovascular changes) and often do not clearly define the region affected. Numerous in vivo studies have indicated that hyperglycemia augments ischemic brain damage; this effect is often attributed to lactic acidosis. To separate the effects on neuronal tissue of ischemia from those due to actions on the cerebrovascular system, we used an in vitro blood-free system as an ischemic model. In our study we evaluated the effects of various combinations of oxygen and glucose levels on evoked synaptic activity in the CA1 region of the rat hippocampal slice preparation. A 50% inhibitory dose for both oxygen and glucose on neuronal synaptic function was determined. It is our intention to use this model for preliminary screening of antihypoxic/anti-ischemic drugs.

Lactic acidosis and recovery of neuronal function following cerebral hypoxia in vitro.

The rat hippocampal slice preparation was used to study the combined effects of hypoxia and lactic acidosis on neuronal function. Control slices were exposed to a standard hypoxic insult while being perfused with normal artificial cerebrospinal fluid (ACSF). Experimental slices were perfused with ACSF containing 1.0, 2.0, 10.0 or 20.0 mM lactic acid, 30 min before and during the same standard hypoxic insult. Following at 30-min recovery period the ability of these slices to respond to orthodromic stimulation by displaying a population spike (synaptic function) was tested. No significant decreases in the recovery rate of synaptic function were found between control and experimental groups, excluding the combination of 20 mM lactic acid and 10 min hypoxia, where such a decrease was found. The combination of 10 mM lactic acid and 12 min hypoxia brought about an increase in the recovery rate of synaptic function. Thus, the adverse effects attributed to lactic acid in vivo were not seen in the present in vitro study. Neuronal tissue appears to be able to handle excess lactic acid by yet, unknown mechanism (high intracellular buffer capacity?). The suggested in vivo damage due to lactic acidosis could originate in the cerebrovascular system. On the other hand, the possibility that lactic acidosis is harmless under hypoxic conditions should also be considered.

Activity of opioid peptidergic system in acupuncture analgesia.

To study the relationship between the opioid peptidergic system (OPS) and acupuncture analgesia (AA), observations have been made both at presynaptic levels and receptor sites in our college since 1975. It was found that AA could be reversed by naloxone in rabbits and in man. The opioid peptide activity increased in human cerebrospinal fluid and in the perfusate from certain brain areas during AA, as revealed by radio-receptor assay. The results indicate that OPS takes an active part in AA. To study further the activity of OPS, experiments were done at the level of nerve cells on rabbits's central gray (PAG). By using multimicropipettes for extracellular recording and iontophoresis of drugs, it was found that opiates produced a naloxone reversible inhibition on the spontaneous discharge of certain neurones. Such neurones were distributed mostly in the ventral part of the PAG. Similar to iontophoretic opiates, electroacupuncture (EA) had an inhibitory effect on PAG neurones and the inhibition could be reversed by iontophoretic naloxone. A correlation existed between the effects of EA and opiates (P less than 0.0174). EA and opiates also showed similar inhibitory effect on nociceptive response of PAG neurones. It was postulated that acupuncture signals activate the brain OPS to exert a double control over the transmission of pain sensation in the PAG, i.e., to block the conveyance of nociceptive impulses at situ and at other relay stations through inhibitory systems. In addition to the PAG, most brain areas important in AA are interrelated to OPS, and the interaction between opioid peptides and opiate receptors in these areas perform an analgesic function as a whole.