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.

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.

Osteopontin is involved in the initiation of cutaneous contact hypersensitivity by inducing Langerhans and dendritic cell migration to lymph nodes.

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and alphav integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.

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.

An essential role for CD44 variant isoforms in epidermal Langerhans cell and blood dendritic cell function.

Upon antigen contact, epidermal Langerhans cells (LC) and dendritic cells (DC) leave peripheral organs and home to lymph nodes via the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Since splice variants of CD44 promote metastasis of certain tumors to lymph nodes, we explored the expression of CD44 proteins on migrating LC and DC. We show that upon antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6, and v9. Antibodies against CD44 epitopes inhibit the emigration of LC from the epidermis, prevent binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.

Telemetered recording of hormone effects on hippocampal neurons.

Frequency-modulated telemetry was used to record the effects of hormones on single-unit activity in the brains of freely moving rats. Corticosterone decreased unit activity in the dorsal hippocampus. Adrenocorticotrophic hormone had the opposite effect.

Stress-induced suppression of immunity in adrenalectomized rats.

Stress-induced suppression of lymphocyte stimulation by phytohemagglutinin was demonstrated in Isolated lymphocytes and in cultures of whole blood from adrenalectomized rats. The results demonstrate that corticosteroid independent mechanisms participate in the suppression of lymphocyte function by stressors. Stress-induced lymphopenia, however, was found to be adrenal dependent, indicating that the modulation of immunity by stress is complex and multidetermined.

Pituitary-adrenal influences on fear responding.

In a passive avoidance situation, hypophysectomized male rats show less fear than normal rats, whereas adrenalectomized rats show greater fear than normals. These results probably occur because hypophysectomized rats lack adrenocorticotrophic hormone, which increases arousal or emotionality, whereas adrenalectomized animals lack certain adrenal steroids, which inhibit excitatory effects. The results indicate that adrenocorticotrophic hormone and certain adrenal steroids have opposite effects in regulating fear-motivated behavior.

Suppression of immunity by stress: effect of a graded series of stressors on lymphocyte stimulation in the rat.

In rats a graded series of stressors produced progressively greater suppression of lymphocyte function, as measured by the number of circulating lymphocytes and by phytohemagglutinin stimulation of lymphocytes in whole blood and isolated cultures. This evidence suggests that stress suppresses immunity in proportion to the intensity of the stressor.

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).

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.

[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.

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.

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.

Evidence for involvement of B lymphocytes in the surveillance of lung metastasis in the rat.

These studies examined the composition of lymphocytes within the lung after the introduction of tumor cells that metastasize to the lung in rats. i.v. delivery of MADB106 tumor cells into syngeneic Fischer 344 rats caused dose- and time-dependent development of lung tumors, with surface metastases evident 7 days after injection and markedly increased 11 days after injection. The total number of lymphocytes recovered from the lung was increased 11 days after injection but not 7 days after injection. When lymphocytes from the lung, spleen, and blood were subjected to fluorescence-activated cell sorting analysis, the most conspicuous change was an increase in the percentage of CD45RA+ cells (i.e., B lymphocytes in the rat) in the lung, with no changes seen in the percentage of natural killer (NKR-P1+), CD4+, or CD8+ cells in the lung. Analysis of the time course showed that B lymphocytes increased in the lung soon after i.v. tumor injection, with an initial peak seen 6 h after injection. Rapid influx of B lymphocytes into lung after i.v. tumor cell injection was also observed in another syngeneic tumor model, i.e., after injection of CC531 cells into WAG rats. To determine whether the influx of B lymphocytes into the lung might participate in tumor surveillance, a high dose of antibody (100 microg) to rat B lymphocytes was given to immunoneutralize these cells; this produced an increase in lung tumors in both models. Finally, Fischer 344 rats were given a s.c. injection of MADB106 tumor cells that made them resistant to lung tumors when given a later i.v. injection of these tumor cells. These animals were found to have an elevated level of B lymphocytes residing in the lung associated with the resistance to lung tumor. These findings suggest that early responses of B lymphocytes are important in protection against tumor development in two rat models of cancer.

The subdiaphragmatic vagus nerves mediate activation of locus coeruleus neurons by peripherally administered microbial substances.

Our earlier studies demonstrated that representative microbial substances--lipopolysaccharide, peptidoglycan, and poly-inosine: poly-cytosine (poly(I):(C))--increased the spontaneous discharge rates and sensory-evoked responses of isolated locus coeruleus (LC) neurons in a dose- and time-related manner after i.p. injection into rats. We then turned our attention to the mechanism by which microbial substances administered into the peritoneal cavity affect the LC neurons. The involvement of the subdiaphragmatic vagus nerves was examined in this regard since several brain responses to peripherally administered lipopolysaccharide have been found to depend upon the integrity of these nerves. The experiments reported here show that lipopolysaccharide, peptidoglycan, and poly(I):(C) all failed to excite LC neurons after i.p. injection into rats that had previously been subjected to complete transection of the subdiaphragmatic vagus nerves. Furthermore, selective transection of the subdiaphragmatic vagus nerve trunks indicated that the dorsal trunk, and not the ventral trunk, was necessary to excite LC neurons in response to i.p. lipopolysaccharide. The inability of LC neurons to respond to i.p. lipopolysaccharide in vagotomized rats is unlikely to be attributed to a desensitization of the neurons to lipopolysaccharide since i.c.v. injection of lipopolysaccharide excited LC neurons in vagotomized rats as it did in vagus-intact rats. These findings suggest that a variety of microbial substances excited LC neurons after administration into the peritoneal cavity in a manner involving the subdiaphragmatic vagus nerves.