Cytisine for smoking cessation: A 40-day treatment with an induction period.

INTRODUCTION: Cytisine is a smoking cessation drug now used worldwide. Most of the data available in the literature predict a 25-day treatment, accepted on the basis of previous clinical experience in Eastern Europe. There are few studies on dosing, and only recently some researchers have tried a longer treatment period. METHODS: This real-world retrospective cross-sectional study analyzed data collected consecutively from 2015 to 2021, in seven smoking cessation centers in north-central Italy. The aim of this study is to evaluate the effectiveness and tolerability of a 40-day cytisine treatment with an induction phase and a slower reduction schedule. Data were collected from a group of 871 patients treated with cysteine, varenicline, and nicotine replacement therapy (NRT). The sample was not randomized. Behavioral support (4-6 sessions, each lasting 20-30 min, plus the evaluation session) was delivered to all patients. RESULTS: Subgroups taking cytisine (n=543 for 40 days), varenicline (n=281 for 12 weeks), and NRT (n=47 for eight weeks) showed biochemically confirmed smoking abstinence at 6 months of 50.5%, 55.9%, and 51.0%, respectively, with a statistically significant difference between cytisine versus varenicline (p<0.01) but not between cytisine versus NRT (p=0.5597). Adverse events were 4.4% with cytisine and 33.3% with varenicline. Behavioral support was an important factor in effectiveness. CONCLUSIONS: This study produced preliminary evidence that the 40-day regimen of cytisine, appears to have less effectiveness in comparison to varenicline but the magnitude of the effect is comparable. The results and tolerability seem to be better than in most other studies.

Personalized and targeted mutational analysis of multiple second primary melanomas under kinase inhibitors.

BACKGROUND: Second primary melanomas (SPMs) are new developed primary melanomas occurring in a subset of patients affected by BRAF-mutated metastatic melanoma during treatment with BRAF-inhibitors. A drug-induced paradoxical activation of mitogen-activated protein kinase (MAPK) signaling pathway in BRAF-wild type/RAS-mutated cells have been proposed as a possible molecular mechanism but data on the mutational status of SPMs are lacking. In order to better understand genetic alterations affecting the biological mechanism of SPMs, we performed a personalized and targeted next-generation sequencing analysis of a patient affected by metastatic melanoma who developed multiple SPMs during treatment with encorafenib (LGX818). METHODS: Using a cancer panel of 50 genes for solid tumors enriched with a custom panel of 10 genes specifically involved in melanoma pathogenesis, we analyzed the primary melanoma, two SPMs, one benign compound nevus and the normal DNA extracted from blood lymphocytes of the patient. RESULTS: We identified HRAS Q61 somatic mutation in one SPM developed in a pre-existing nevus. In the primary melanoma, besides the BRAF mutation, we identified the clinically actionable IDH1 R132C somatic mutation. Both SPMs were BRAF wild type. The patient harbors the recently recognized pathogenetic germline variant KDR Q472. We observed that mutations detected in tumor samples involving genes related to melanoma pathogenesis (TP53, PIK3CA, FGFR3, ATF1, KIT, HRAS and MAP2K2) were present in heterozygosis in the germline status of the patient. CONCLUSIONS: Our results support the paradoxical mechanism of MAPK pathway for SPMs under BRAF inhibitors. Moreover, they suggest that targeted mutational assessment based on matching somatic and germline analysis represent a promising approach to detect the neoplastic landscape of the tumor and to identify most accurate treatment in metastatic melanoma patient.

Seriate cytology vs molecular analysis of peritoneal washing to improve gastric cancer cells detection.

BACKGROUND: Intraperitoneal malignant cells detection in patients with gastric cancer is associated with a significant decrease in overall survival. The overall accuracy of cytological examination of peritoneal lavages, however, is quite low, and intraperitoneal recurrence has been observed even in patients with negative cytology. Immunocytochemistry and molecular techniques have been investigated to improve high-risk patients' identification with variable results. The aim of this study was to compare the performance of different laboratory methods applied to peritoneal washing, to improve the cytological identification of malignant cells. METHODS: We prospectively evaluated 21 patients who underwent surgery and peritoneal lavage for gastric cancer. Among them, 18 had negative cytology and three were positive for malignant cells. For each patient, immunohistochemistry with BerEP4 antibody was performed on seriate sections of cellblock preparation at different levels, using the method reported for sentinel nodes in other types of cancer. Paired frozen quotes of washing fluids were evaluated by qRT-PCR with primer for mRNA of Ceacam5. RESULTS: In 4 of 18 patients with previous negative routine cytology, isolated neoplastic cells in seriate sections of the cellblock inclusion have been found. Results showed to be coherent with molecular analysis for CEA mRNA. CONCLUSION: The sensitivity and specificity of peritoneal washing analyses should be notably improved by immunohistochemistry applied to multilevel cellblock sectioning. The method is less expensive and more widely applicable than molecular analysis, in each laboratory setting. This approach allows detection of minimum peritoneal seeding in patients with gastric cancer.

Association between osteoprotegerin G1181C and T245G polymorphisms and diabetic charcot neuroarthropathy: a case-control study.

OBJECTIVE: Charcot neuroarthropathy is a disabling complication of diabetes. Although its pathogenesis remains unknown, we suppose that genetics may play a relevant role. RESEARCH DESIGN AND METHODS: We performed a case-control study with 59 subjects with diabetic Charcot neuroarthropathy (Ch group), 41 with diabetic neuropathy without Charcot neuroarthropathy (ND group), and 103 healthy control subjects (H group) to evaluate the impact of two single nucleotide polymorphisms (SNPs) of the osteoprotegerin gene (G1181C and T245G) on the risk of Charcot neuroarthropathy. RESULTS: Regarding the SNPs of G1181C, we found a significant linkage between the G allele and Charcot neuroarthropathy (Ch vs. ND, odds ratio [OR] 2.32 [95% CI 1.3-4.1], P = 0.006; Ch vs. H, 2.10 [1.3-3.3], P = 0.002; and ND vs. H, 0.90 [0.7-1.9], P = 0.452); similarly, we found a linkage with the G allele of T245G (Ch vs. ND, 6.25 [2.2-19.7], P < 0.001; Ch vs. H, 3.56 [1.9-6.7], P = 0.001; and ND vs. H, 0.54 [0.6-5.7], P = 0.304), supporting a protective role for the allele C and T, respectively. For this reason we investigated the frequency of the protective double homozygosis CC + TT (7% in Ch) that was significantly lower in Ch compared with H (0.18 [0.06-0.5], P = 0.002) and with ND (0.17 [0.05-0.58], P = 0.006), whereas there was no difference between H and ND (1.05 [0.43-2.0], P = 0.468). In a multivariate logistic backward regression model, only weight and the lack of CC and TT genotypes were independently associated with the presence of Charcot neuroarthropathy. CONCLUSIONS: This is the first study that shows an association between genetic regulation of bone remodeling and Charcot neuroarthropathy.

Topiramate and triptans revert chronic migraine with medication overuse to episodic migraine.

OBJECTIVE: This is a randomized, double-blind versus placebo study aimed at evaluating the efficacy of topiramate (TPM) in reducing the number of days with headache and the amount of acute medication taken monthly in patients with chronic migraine with medication overuse. We also studied the efficacy of single triptans available in Italy in interrupting headache crises during preventive treatment. METHODS: The studied sample was made up of 50 subjects: 30 patients were randomized for treatment with TPM, 100 mg/d, and 20 for placebo. Subjects treated with TPM were further randomized to evaluate, in double-blind versus placebo, the efficacy of single triptans available in Italy. The double-blind phase consisted of a titration phase (4 weeks) and of a maintenance phase (8 weeks). OUTCOME MEASURES: The reduction in the number of days with headache per 28 days and the reduction in the amount of acute medication taken per 28 days throughout the clinical trial in the TPM group were compared with those of the placebo group; the number of patients who were pain-free at 2 hours after the triptan intake and the headache recurrence rate in the 22 hours after the pain-free condition in the triptan group were compared with those of the placebo group. We also looked at tolerability profile. RESULTS: The group treated with TPM had a significant reduction in the number of days with headache (P < 0.0001 vs placebo) and in the mean amount of acute medication taken (P < 0.0001 vs placebo); all triptans were superior to placebo; there were no significant differences between different triptans; the analgesic effect of triptans increased throughout the trial. CONCLUSIONS: Topiramate proved to be well tolerated and effective in reverting chronic migraine with medication overuse to episodic migraine.

Reverse transcriptase-PCR analysis of apoptosis-regulating gene expression in human benign prostatic hyperplasia.

BACKGROUND: To determine whether an imbalanced interaction between proapototic and antiapoptotic signals may account for the loss of the normal cell growth control in benign prostatic hyperplasia (BPH), the expression of some apoptosis-regulating genes (bcl-2, bax, c-myc, fas) was investigated. PATIENTS AND METHODS: BPH specimens were obtained from 20 patients who underwent trans-urethral resection of the prostate (TURP) or adenomectomy. Gene expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and its correlation with age and serum PSA level was also investigated. RESULTS: Genes were found to be differentially expressed in BPH tissues. In particular, the antiapoptotic gene bcl-2, which was found in 18/20 samples, gave the weakest signal (p < 0.05 - p < 0.001, Wilcoxon's signed rank test), whereas the cell cycle regulator c-myc was detected in all the specimens and was the most highly expressed (p < 0.001). A positive relationship between the expression of bcl-2 and that of the two proapoptotic genes bax and fas was observed (p < 0.05, Spearman's rank correlation test), as well as between c-myc and fas levels (p < 0.005). Moreover, bax expression positively correlated with age and PSA (p < 0.02), which have also been shown to directly correlate (p < 0.01). CONCLUSION: The higher expression of the oncogene c-myc suggests the activation of mitogenic signals within hyperplastic prostate tissue which a relatively high expression of the proapoptotic genes bax and fas fails to counterbalance.

Human hepatocytes in mice receiving pre-immune injection with human cord blood cells.

It is well established that certain subpopulations of human adult stem cells can generate hepatocyte-like cells when transplanted into adult immunosuppressed mice. In the present study, we wanted to explore whether xeno-transplantation of human cord blood CD34(+) (hCBCD34(+)) cells during pre-immune stages of development in immunocompetent mice might also lead to human-mouse liver chimerism. Freshly isolated hCBCD34(+) cells were xeno-transplanted into non-immunosuppressed mice by both intra-blastocyst and intra-fetal injections. One and four weeks after birth, immunostaining for different human-specific hepatocyte markers: human hepatocyte-specific antigen, human serum albumin, and human alpha-1-antitrypsin indicated the presence of human hepatocyte-like cells in the livers of transplanted animals. Detection of human albumin mRNA further corroborated the development of pre-immune human-mouse chimeras. The current report, besides providing new evidence of the potential of hCBCD34(+) cells to generate human hepatocyte-like cells, suggests novel strategies for generating immunocompetent mice harboring humanized liver.

Apoptosis-related gene expression affected by a GnRH analogue without induction of programmed cell death in LNCaP cells.

BACKGROUND: In this study we confirmed the ability of a Gonadotropin Releasing Hormone (GnRH) agonist, leuprorelin acetate (LA), to counteract or even suppress the 5alpha-dihydrotestosterone (DHT)-stimulated growth of androgen-sensitive prostate cancer cells (LNCaP). Since the cellular mechanisms mediating this effect are not well defined, we investigated the activity of LA, also in combination with DHT or with cyproterone acetate (CA), on the expression of genes (bcl-2, bax, c-myc) which may contribute to the proliferative behaviour of LNCaP cells. In addition, experiments aimed to evaluate the action of the analogue on apoptosis were performed. MATERIALS AND METHODS: Gene expression was evaluated by RT-PCR and Western blotting on cells treated with LA (10(-11) or 10(-6) M), alone or combined with 10(-9) M DHT or 10(-7) M CA. The occurrence of apoptosis following treatment with LA (10(-11), 10(-6) or 10(-5) M), alone or combined with 10(-9) M DHT, was assessed by DNA fragmentation analysis. RESULTS: Both the mRNA and protein of the anti-apoptotic gene bcl-2 were induced (30-125%) by DHT after 24-144 h. LA decreased bcl-2 mRNA (10-40%), while it did not unequivocally affect protein expression. The analogue always reduced (13-74%) both mRNA and protein levels obtained under DHT treatment. The mRNA and protein of the pro-apoptotic gene bax were down-regulated by DHT (15-40%), while LA generally induced bax protein but not its mRNA. LA counteracted DHT activity, even increasing bax protein levels over the controls. c-myc mRNA and protein were enhanced by DHT (15-45%) but down-regulated by LA (10-40%). Once more, the androgen effect was antagonized by LA, sometimes reducing c-myc content below the controls. CA produced the most similar effects to those triggered by DHT. The hormonal treatment did not induce any DNA fragmentation. CONCLUSION: In spite of gene modulation, apoptosis was not observed under LA treatment, in agreement with the lack of a cell growth effect when the analogue was used alone. Nevertheless, the observed changes in gene expression may be directly or indirectly involved in the antiproliferative effect of LA on androgen-stimulated cells.

The intracoelomic route: a new approach for in utero human cord blood stem cell transplantation.

The intracoelomic route for in utero hematopoietic stem cell transplantation has been evaluated in pre-immune fetal sheep and the engraftment characteristics defined. Twelve ovine fetuses (gestational ages: 40-45 days) received intracoelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspension of the liver, spleen, bone marrow and thymus by flow cytometry, cloning assays and polymerase chain reaction (PCR) analysis for human beta(2)-microglobulin gene. The engraftment of liver samples was also evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH) and immunohistochemistry. Four fetuses (33%) aborted shortly after intracoelomic transplantation and were not evaluable for engraftment. Engraftment was detected in 4 fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degree of engraftment in these 4 fetuses ranged from 6 to 22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (No. 435184) and 105 (Nos 915293, 037568) days and 1 fetus delivered at term, which received CD34-selected cord blood cells, had human engraftment with 10, 32, 20 and 10% CD45+ cells in bone marrow, respectively. A further check of human chimerism was done at 1 year after birth of the fetus delivered at term and 7.6% of bone marrow chimerism was detected. In 6 out of 8 fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta(2)-microglobulin which also identified human cells in brain, spinal cord, heart, lung and skeletal muscle. On liver samples, FISH and RT-PCR confirmed the xenograft of human cells and the immunohistochemical analysis detected human markers of hematopoietic and hepatic lineage of differentiation. This preliminary study indicates that intracoelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.

A novel route of transplantation of human cord blood stem cells in preimmune fetal sheep: the intracelomic cavity.

The intracelomic route for in utero hematopoietic stem cell transplantation was evaluated in preimmune fetal sheep and the engraftment characteristics were defined. Twelve twin ovine fetuses (gestational age: 40-45 days) received intracelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspensions of the liver, spleen, bone marrow, and thymus by flow cytometry, cloning assays, and polymerase chain reaction (PCR) analyses of human beta2-microglobulin. Four fetuses (33%) aborted shortly after intracelomic transplantation and were not evaluable for engraftment. Engraftment was detected in four fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degrees of engraftment in these four fetuses ranged from 6%-22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (no. 435184) and 105 (no. 915293, no. 037568) days and one fetus delivered at term that received CD34-selected cord blood cells had human engraftment with 10%, 32%, 20%, and 10% CD45(+) cells in bone marrow, respectively. In six of eight fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta2-microglobulin, which also identified human cells in brain, spinal cord, heart, lung, and skeletal muscle. This preliminary study indicates that intracelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.

Regulation of prostate-specific antigen gene expression by an LH-RH analogue in human prostatic cells.

BACKGROUND: We investigated the regulation of prostate-specific antigen (PSA) gene expression in prostatic tumor cells by an LH-RH analogue, Leuprorelin acetate (LH-RHa), alone or combined with dihydrotestosterone (DHT). MATERIALS AND METHODS: PSA gene expression was evaluated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. RESULTS: LH-RHa at both high and low concentrations induced a diminution in the levels of PSA gene expression which reached 70% in androgen-insensitive PC-3 cells. The analogue lowered the levels of expression obtained under DHT stimulation in LNCaP cells. These effects were visible from 6-24 hours of treatment and persisted until 120 hours. CONCLUSION: PSA gene expression is directly regulated by LH-RHa in both LNCaP and PC-3 cells. High concentrations of the analogue are particularly effective, but low doses of the drug have a similar behaviour. Interestingly, Leuprorelin acetate is able to counteract the androgen-induced gene expression. Our results suggest that, at least in androgen-sensitive cells, the analogue may regulate PSA gene expression also by interfering with the androgen receptor machinery.

Human milk contains S100B protein.

The present study constitutes the first finding of the calcium-binding protein S100B and of its mRNA in human milk, as revealed by a quantitative immunoluminometric assay, by Western blot analysis and by reverse transcription-polymerase chain reaction (RT-PCR) assay followed by restriction enzyme digestion. The concentration of S100B in milk is markedly higher than that observed in other biological fluids such as cord blood, peripheral blood, urine, cerebrospinal fluid and amniotic fluid. This finding could be related to a possible trophic role, which has been hypothesized for the protein.

Cyclooxygenase-2 and caspase 3 expression in trimethyltin-induced apoptosis in the mouse hippocampus.

The neurotoxicant trimethyltin (TMT) induces massive neuronal loss in vivo in the hippocampus of rodents, accompanied by behavioral alterations. The present study investigates the pattern of cell death after in vivo administration of TMT to adult mice. In the granular cell layer of the Dentate Gyrus, TUNEL staining detected DNA fragmentation, and apoptotic bodies were also evident. In addition, a ladder pattern of internucleosomal DNA fragmentation was shown in agarose gel electrophoresis. We show that activated caspase-3, which is known to play a pivotal role in apoptotic processes, is clearly expressed by degenerating neurons. Inducible cyclooxygenase is also expressed at cytoplasmic level by degenerating granular neurons, suggesting that this enzyme may participate in TMT-induced neurodegeneration.