Myelopreservation with the CDK4/6 inhibitor trilaciclib in patients with small-cell lung cancer receiving first-line chemotherapy: a phase Ib/randomized phase II trial.

BACKGROUND: Chemotherapy-induced damage of hematopoietic stem and progenitor cells (HSPC) causes multi-lineage myelosuppression. Trilaciclib is an intravenous CDK4/6 inhibitor in development to proactively preserve HSPC and immune system function during chemotherapy (myelopreservation). Preclinically, trilaciclib transiently maintains HSPC in G1 arrest and protects them from chemotherapy damage, leading to faster hematopoietic recovery and enhanced antitumor immunity. PATIENTS AND METHODS: This was a phase Ib (open-label, dose-finding) and phase II (randomized, double-blind placebo-controlled) study of the safety, efficacy and PK of trilaciclib in combination with etoposide/carboplatin (E/P) therapy for treatment-naive extensive-stage small-cell lung cancer patients. Patients received trilaciclib or placebo before E/P on days 1-3 of each cycle. Select end points were prespecified to assess the effect of trilaciclib on myelosuppression and antitumor efficacy. RESULTS: A total of 122 patients were enrolled, with 19 patients in part 1 and 75 patients in part 2 receiving study drug. Improvements were seen with trilaciclib in neutrophil, RBC (red blood cell) and lymphocyte measures. Safety on trilaciclib+E/P was improved with fewer ≥G3 adverse events (AEs) in trilaciclib (50%) versus placebo (83.8%), primarily due to less hematological toxicity. No trilaciclib-related ≥G3 AEs occurred. Antitumor efficacy assessment for trilaciclib versus placebo, respectively, showed: ORR (66.7% versus 56.8%, P = 0.3831); median PFS [6.2 versus 5.0 m; hazard ratio (HR) 0.71; P = 0.1695]; and OS (10.9 versus 10.6 m; HR 0.87; P = 0.6107). CONCLUSION: Trilaciclib demonstrated an improvement in the patient's tolerability of chemotherapy as shown by myelopreservation across multiple hematopoietic lineages resulting in fewer supportive care interventions and dose reductions, improved safety profile, and no detriment to antitumor efficacy. These data demonstrate strong proof-of-concept for trilaciclib's myelopreservation benefits. CLINICAL TRAIL NUMBER: NCT02499770.

Cats' Internal Exposure to Selected Brominated Flame Retardants and Organochlorines Correlated to House Dust and Cat Food.

Pet cats may be used as a biomarker for assessing exposures to organohalogen compounds (OHCs) adsorbed to household dust in home environments. This study explores two exposure routes of OHCs, ingestion of OHCs (i) via house dust and (ii) via cat food. House dust from 17 Swedish homes and serum from the participating families' pet cats were collected, and cat food was purchased matching the diet reported. Paired samples of cat serum, house dust, and cat food were analyzed for brominated flame retardants/natural products (polybrominated diphenyl ethers (PBDEs), decabromobiphenyl (BB-209), decabromodiphenyl ethane (DBDPE), 2,4,6-tribromophenol (2,4,6-TBP), OH-PBDEs) and organochlorines (polychlorinated biphenyls (PCBs), 1,1-bis(4,4'-dichlorodiphenyl)-2,2,2-trichloroethane (4,4'-DDT), 1,1-bis(4,4'-dichlorodiphenyl)-2,2-dichloroethene (4,4'-DDE), hexachlorobenzene (HCB), pentachlorophenol (PCP)). Significant correlations were found between serum and dust samples from the living rooms for BDE-47 (p < 0.035), BDE-99 (p < 0.035), and BDE-153 (p < 0.039), from the adult's bedroom for BDE-99 (p < 0.019) and from all rooms for BDE-99 (p < 0.020) and BB-209 (p < 0.048). This is the first time a correlation between cat serum levels and household dust has been established, a finding that supports the hypothesis that dust is a significant exposure route for cats. Serum levels were also significantly correlated with concentrations found in cat food for 6-OH-BDE47 (p < 0.002), 2,4,6-TBP (p < 0.035), and BB-209 (p < 0.007). DBDPE was found in high concentrations in all dust (median 154 pmol/g) and food samples (median 0.7 pmol/g lw) but was below detection in serum samples, suggesting low or no bioavailability for DBDPE in cats.

Mice with heterozygous deficiency of manganese superoxide dismutase (SOD2) have a skin immune system with features of "inflamm-aging".

Dendritic cells (DC) are central in regulating skin immunity. Immunosenescence is associated with a chronic inflammatory state. Little is known about the contribution of DC to "inflamm-aging". When determining langerhans cell (LC) numbers, we found a 60 % reduction of LC in aged epidermis. Reactive oxygen species(ROS) are linked with aging. The mitochondrial manganese superoxide dismutase (SOD2) is in the first line of antioxidant defense. We investigated the function of DC from SOD2 heterozygous mice (SOD2+/-) and found that at 4 months of age LC numbers are not altered, but activated LC have impaired expression of MHC-II and CD44. Immature SOD2+/- DC produced increased proinflammatory IL-6 and chemokines CXCL1 and CXCL2. Upon challenge SOD2+/- DC accumulated ROS. When activating SOD2+/- DC by LPS they less efficiently upregulated MHC-II, CD86 and CD44. Surprisingly, in vivo contact hypersensitivity (CHS) was enhanced in SOD2+/- mice although SOD2+/- DC were less potent in stimulating wt T cells. However, SOD2+/- T cells showed increased proliferation, even when stimulated with SOD2+/- DC, possibly explaining the increased CHS. Our findings suggest that SOD2 is a molecular candidate in the regulation of "inflamm-aging" conveying both immunosuppressive and proinflammatory signals through alteration of DC and T cell functions.

Colon Cancer Staging in Vulnerable Older Adults: Adherence to National Guidelines and Impact on Survival.

BACKGROUND: There is concern that elders are not adequately evaluated prior to colon cancer surgery. We sought to determine adherence with ACOVE-3 (Assessing Care of Vulnerable Elders) quality indicators for pre-operative staging prior to colectomy for colon cancer utilizing the Surveillance, Epidemiology and End Results (SEER)-Medicare linked database (1992-2005). METHODS: We determined the proportion of patients aged 75 and older who had preoperative staging prior to colectomy for colon adenocarcinoma. Preoperative staging was defined as abdominopelvic computed tomography or magnetic resonance imaging scan (SCAN) and colonoscopy or flexible sigmoidoscopy (SCOPE). Multivariate logistic regression identified predictors of adherence. Odds ratios were adjusted for comorbidity, socioeconomic status, and disease severity. The association of adherence to ACOVE-3 and survival was quantified. RESULTS: Of the 37,862 patients, the majority were 75-84 years, 28% of the patients were ≥85 years. Regarding preoperative staging in the 6-month interval prior to surgical resection, 8% had neither SCAN nor SCOPE, 6% had only SCAN, 43% had only SCOPE, and 43% had both SCAN and SCOPE. Compared to patients who were not staged, those evaluated with either SCOPE alone or SCAN plus SCOPE had lower odds of 3-year mortality. Patients who were staged with SCAN alone had an increased odds of death compared to those who had neither SCAN or SCOPE. CONCLUSIONS: These data demonstrate that the majority of vulnerable elders with colon cancer did not receive appropriate preoperative staging prior to resection. The findings also confirm that adherence to ACOVE-3 guidelines is associated with improved long-term survival.

The thymus in autoimmune Myasthenia Gravis: Paradigm for a tertiary lymphoid organ.

In autoimmune Myasthenia Gravis (MG), a neuromuscular disease generally mediated by autoantibodies against the acetylcholine receptor (AChR), the muscle is the target organ of the autoimmune attack, while the thymus seems to be the primary production site of the autoantibodies. In the majority of patients with anti-AChR antibodies, it is characterized by the presence of germinal centers, which contain B cells that produce anti-AChR antibodies. In this review, we summarize recent results regarding neoangiogenic processes, cell infiltration and modified chemokine expression in the MG thymus, which are typical features of secondary lymphoid organs. The structural and functional changes in the MG thymus therefore allow us to declare it to be an archetype for tertiary lymphoid neogenesis providing optimal settings for the interaction between lymphocytes and antigen presenting cells in order to elicit an immune response. We further discuss factors that may have a key role in the transformation of the MG thymus into a tertiary lymphoid organ, such as IFN type I and dsRNA signaling. These factors could also be of importance in other autoimmune diseases, especially those characterized by tertiary lymphoid neogenesis.

Adipocyte fatty acid-binding protein as a novel prognostic factor in obese breast cancer patients.

Several adipocytokines, such as leptin or adiponectin, are associated with obesity and the risk for breast cancer. Adiopcyte fatty acid binding-protein(A-FABP) is another protein found in adipose tissue;therefore, we investigated the association of A-FABP with the occurrence and prognosis of breast cancer. In our study,200 women attending the University of Ulm for breast surgery between the years 2005 and 2007 were included;159 had histologically confirmed breast cancer; 41 had histologically confirmed benign lesions. Serum levels ofA-FABP, leptin, and adiponectin were measured, and their relationship to body-mass-index (BMI), breast cancer, and tumor characteristics were analyzed; logistic regression model was adjusted to age, BMI, menopausal status, use of Hormone Replacement Therapy (HRT), and family history of breast cancer. Serum A-FABP levels were found to be significantly higher in obese (BMI C 25) than in non-obese women (BMI B 24.9), 41.16 ng/ml and 24.95 ng/ml,respectively (P\0.0001). Independent of obesity, the serum A-FABP levels were significantly higher in breast cancer patients (34.65 ng/ml) than in healthy controls(24.47 ng/ml), P\0.0001; the odds ratio (1.038, P\0.05,95% confidence interval 1.001-1.72) showed a significant association of A-FABP with breast cancer risk. Serum leptin levels showed a strong correlation with BMI(rs = 0.78) and were significantly higher in breast cancer patients (20.87 ng/ml) than in controls (14.90 ng/ml),P\0.05. In contrast, adiponectin showed no significant association with breast cancer. Concerning tumor characteristics,A-FABP was positively connected with tumor size (T C 2 cm, P\0.05) and nodal-status (P\0.05).Our study reveals that high A-FABP serum levels are associated with obesity, breast cancer risk, and adverse tumor characteristics.

Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation.

Cryopreservation as a process can be divided into two methods: conventional freezing and vitrification. The high effectiveness of vitrification in comparison with conventional freezing for human oocytes and embryos is shown, whereas data on human ovarian tissue are limited. The aim of this study was to compare the safety and effectiveness of conventional freezing and vitrification of human ovarian tissue. Ovarian tissue fragments from 15 patients were transported to the laboratory within 22-25 h in a special, isolated transport box that can maintain a stable temperature of between 5 and 8 degrees C for 36 h. Small pieces of ovarian tissue (0.3-1 x 1-1.5 x 0.7-1 mm) were randomly distributed into three groups: group 1, fresh pieces immediately after receiving transport box (control); group 2, pieces after vitrification; and group 3, pieces after conventional freezing. After thawing, all the pieces were cultured in vitro. The viability and proliferative capacity of the tissue by in vitro production of hormones, development of follicles, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression after culture were evaluated. A difference between freezing and vitrification was not found in respect to hormonal activity and follicle quality. The supernatants showed 17-beta estradiol concentrations of 365, 285, and 300 pg/ml respectively, and progesterone concentrations of 3.82, 1.99, and 1.95 ng/ml respectively. It was detected that 95, 80, and 83% follicles respectively were morphologically normal. The molecular biological analysis, however, demonstrated that the GAPDH gene expression in ovarian tissue after vitrification was dramatically decreased in contrast to conventional freezing. For cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification, because of higher developmental potential.

Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose.

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 mul suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37 degrees C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5',6,6'-tetrachloro-1-1',3,3'-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1+/-3.2%, P<0.05) when compared with controls (19.4+/-1.9%). The combination of HSA and sucrose (65.2+/-2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6+/-4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.

Rats selectively-bred for behavior related to affective disorders: proclivity for intake of alcohol and drugs of abuse, and measures of brain monoamines.

Several lines of rats potentially useful for studying affective disorders have been developed in our laboratory though selective breeding for behavioral characteristics. The propensity of these lines to consume alcohol and other drugs of abuse (amphetamine and cocaine) was examined. Also, measurement of the concentration of brain monoamines - norepinephrine, dopamine, and serotonin - as well as estimation of their metabolism by measurement of the major extracellular metabolites of these monoamines was carried out to examine possible relationships of brain chemistry to the behavioral characteristics shown by these lines, as well as to their propensity for drug usage. The lines of rats are: Swim Low-active (SwLo) and Swim High-active (SwHi), which show either very low (SwLo) or very high (SwHi) amounts of motor activity in a swim test; Swim-test Susceptible (Susceptible or SUS) and Swim-test Resistant (Resistant or RES), which are highly susceptible (SUS) or highly resistant (RES) to having their swim-test activity depressed by being exposed to a stressful condition prior to the swim test; and Hyperactive (HYPER), which show spontaneous nocturnal hyperactivity compared to non-selectively bred (i.e., normal) rats as well as both extreme hyperactivity and behavioral depression after being exposed to a stressful condition. Regarding alcohol and drug usage, SUS rats readily consume alcohol while all other lines including non-selected, normal rats do not, and SwLo rats show a strong tendency to consume amphetamine and cocaine. Marked differences in brain monoamines were found between the various lines and normal rats, with salient differences seen in norepinephrine, particularly in the hippocampus, and in dopamine in forebrain regions (striatum and nucleus accumbens).

[Pseudo-Kaposi Sarcoma or Kaposi Sarcoma? Kaposi-sarcoma-like skin lesions in a patient with AIDS]. / Pseudo-Kaposi-Sarkom oder Kaposi-Sarkom? Kaposiforme Hautveränderungen bei einem Patienten mit AIDS.

We report on a patient with AIDS stage C3, who received haemodialysis for terminal renal insufficiency and presented with Kaposi sarcoma-like skin lesions on the left hand, distal of his dialysis shunt. Histology, immunohistochemistry and PCR analysis did not support the initially favoured diagnosis of a Kaposi sarcoma, but revealed a pseudo-Kaposi sarcoma related to the Stewart-Bluefarb-syndrome.

Influence of hypo- and hyperglycaemia on plasma leptin concentrations in healthy women and in women with polycystic ovary syndrome.

BACKGROUND: Insulin resistance and obesity play an important role in the pathogenesis of polycystic ovary syndrome (PCOS). It is known that experimentally induced insulin resistance diminishes the stimulatory effect of insulin on leptin secretion. It is not yet known whether the long-term insulin resistance as found in PCOS patients alters the leptin response to hypo- and hyperglycaemia. METHODS: We induced hyper- and hypoglycaemia by glucose clamp technique in 7 patients with PCOS and 20 healthy controls. After a plasma glucose level of 8.8 mmol/l was reached, the plasma glucose level was reduced stepwise to 6.8, 4.8 and 2.8 mmol/l. RESULTS: The PCOS patients required lower glucose infusion rates to reach the glycaemic targets (P < 0.05). Serum insulin and C-peptide concentrations increased significantly during the clamp compared with the baseline in both groups (P < 0.001 for insulin, and P < 0.001, P < 0.005 for C-peptide control and PCOS, respectively) and increased significantly more in PCOS patients compared with the control group (both P < 0.05). Basal leptin levels were significantly higher in the PCOS group than in the control group (P = 0.005). In the controls, the leptin concentration increased significantly during the clamp (P < 0.001 for each glycaemic target), whereas in the PCOS group, leptin secretion increased only during hypoglycaemia (P = 0.04). CONCLUSIONS: Compared with the healthy controls, the response of leptin secretion to hyper- and hypoglycaemia was diminished in PCOS patients. Changes in leptin secretion seem not to be caused by hyper- and hypoglycaemia, but rather by hyperinsulinaemia. Reduced insulin sensitivity seems to be responsible for the diminished leptin response, which might contribute to the obesity found in PCOS patients.

Regulation of GnRH I receptor gene expression by the GnRH agonist triptorelin, estradiol, and progesterone in the gonadotroph-derived cell line alphaT3-1.

The secretion of luteinizing hormone (LH) and the GnRH receptor (GnRH-R) concentration are modulated by ovarian steroids and GnRH. To elucidate whether this regulation is due to alterations at the transcriptional level, we examined the GnRH I-R mRNA expression in the gonadotroph-derived cell line alphaT3-1 treated with different estradiol and progesterone paradigms and the GnRH I agonist triptorelin. alphaT3-1 cells were treated with different steroid paradigms: 1 nM estradiol or 100 nM progesterone for 48 h alone or in combination. Cells were exposed to 10 nM or 100 pM triptorelin for 30 min, 3 h, 9 h, or, in pulsatile way, with a 5-min pulse per hour. The GnRH I-R mRNA was determined by Northern blot analysis. GnRH I-R mRNA from cells treated with continuous triptorelin decreased in a time- and concentration-dependent manner. Pulsatile triptorelin increased GnRH I-R gene expression. Progesterone alone further enhanced this effect, whereas estradiol and its combination with progesterone diminished it. Continuous combined treatment with estradiol and progesterone lead to a significant decrease of GnRH I-R mRNA by 30% and by 35% for estradiol alone. The addition of 10 nM triptorelin for 30 min or 3 h could not influence that steroid effect. In conclusion, estradiol and progesterone exclusively decreased GnRH I-R mRNA in alphaT3-1 cells no matter whether they are treated additionally with the GnRH I agonist triptorelin. The enhanced sensitivity of gonadotrophs and GnRH I-R upregulation by estradiol is not due to increased GnRH I gene expression because GnRH I-R mRNA is downregulated by estradiol and progesterone. Other pathways of the GnRH I-R signal transduction might be involved.

Short-term effects of IGF-I and estradiol on LH secretion from female rat gonadotrophs.

OBJECTIVE: Growth factors and ovarian steroids modulate LH-secretion from pituitary gonadotrophs. Our previous studies demonstrated that long-term IGF-I treatment enhanced LH-secretion from female rat pituitary cells and estradiol facilitated this effect. The effects of estradiol on LH secretion are time-dependent. Short-term treatment inhibited, long-term treatment enhanced GnRH-induced LH-secretion in serum-containing medium. Here we tested the short-term actions of IGF-I and its interaction with estradiol and whether IGF-I is a prerequisite for the negative effect of short-term estradiol treatment in female rat pituitary cells. DESIGN: Pituitary cells were incubated with a series of increasing concentrations of estradiol (1 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 10 nM and 100 nM) for 4 h, IGF-I (10 pM, 100 pM, 1 nM and 10 nM) for 4 h and 14 h and their combinations for 4h in serum-free medium, and then stimulated with 1 nM GnRH during the last 3h of incubation. To clarify the role of IGF-I, cells were incubated simultaneously with estradiol, IGF-I and antibody against IGF-I. LH was measured by radioimmunoassay. RESULTS: Short-term IGF-I treatment did not modify basal or GnRH-induced LH-secretion. Short-term treatment with estradiol did not affect basal or GnRH-induced LH-secretion in serum-free medium. The addition of 100 pM IGF-I to serum-free medium established the negative effect of estradiol short-term treatment on GnRH-induced LH-secretion. The addition of IGF-I antibody fully abolished the negative effect of estradiol. CONCLUSIONS: In conclusion, effects of IGF-I on LH-secretion in female rat pituitary cells require long-term treatment. The negative effect of estradiol short-term treatment on GnRH-induced LH-secretion is dependent on serum-containing medium or the addition of 100 pM IGF-I to serum-free medium.

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells.

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.