Genome-wide CRISPR/Cas9 library screening identified ATM signaling network genes as critical drivers for resistance to ATR inhibition in soft-tissue sarcomas: synthetic lethality and therapeutic implications.

Soft-tissue sarcoma (STS) are a heterogeneous group of rare tumors with different biological behavior that are fatal in more than 40% of cases, due to their metastatic evolution and inadequate treatment options. ATR inhibition already showed an activity, even if modest, in broad pre-clinical models of STS. By using genome-wide CRISPR/Cas9 library screening, we identified ATM signaling network genes as critical drivers for resistance to the specific ATR inhibitor AZD6738. The role of such genes in resistance to AZD6738 was confirmed by using CRISPR/Cas9 knockout models. More strikingly, the ATM inhibitor AZD0156 works synergistically with AZD6738 in vitro and abolishes STS growth in vivo in our models of most frequent histotypes (such as dedifferentiated liposarcoma, leiomyosarcoma, and undifferentiated pleomorphic sarcoma among others). Moreover, the combination of AZD6738 and AZD0156 induced significantly higher levels of DNA damage than either drug used as single agent alone. In summary, our results demonstrate that targeting ATM is an effective approach to overcome resistance to ATR inhibition in different STS subtypes, including the most frequent histologies.

GSK3-beta as a candidate therapeutic target in soft tissue sarcomas.

Soft tissue sarcoma (STS) is a predominantly fatal rare malignancy with inadequate treatment options. Glycogen synthase kinase 3ß (GSK-3ß) is an emerging target in human malignancies. Its therapeutic relevance in STS is unknown. We analyzed the prognostic impact of GSK-3ß gene and protein expression in two independent cohorts of patients with STS. We then treated STS cell lines and mice xenografts with a novel GSK-3 inhibitor 9-ING-41 alone or in combination with chemotherapy. We demonstrated that 9-ING-41 treatment induced significant STS cells apoptosis and was synergistic in vivo when combined with chemotherapy. Mechanistically, 9-ING-41 induces significant apoptosis of STS cells via suppression of NF-κB-mediated X-linked inhibitor of apoptosis protein (XIAP) expression. These data support the inclusion of patients with STS in clinical studies of 9-ING-41 alone and in combination with chemotherapy.

Memory T cells in cancer immunotherapy: which CD8 T-cell population provides the best protection against tumours?

Cancer immunotherapy strategies often fail because of immunosuppressive mechanisms present in the tumour-bearing host. Adoptive T-cell transfer therapy circumvents this problem by activating tumour-specific CD8(+) T cells in vitro and transferring them back into the patient. Classically, effector T cells have been used in these studies because of their potent anti-tumour activity. However, it is becoming apparent that highly activated effector cells may become terminally differentiated, display impaired proliferation and survival in vivo, and mediate short-term anti-tumour effects. In contrast to effector cells, memory cells have enhanced proliferative potential and survival, and the potential to provide more robust and enduring protection against tumours. Here, we discuss key studies in the field of adoptive T-cell transfer, along with some of our own results relating to this area. Based on the body of existing research, it is clear that CD8(+) T cells with memory potential are superior to terminally differentiated effectors in mediating successful tumour clearance. Opinions remain divided as to whether the central memory or effector memory T-cell subset is capable of providing the best protection against tumours. We propose that as these cell types have different but complementary benefits for the anti-tumour immune response, the ideal cell population to use for adoptive T-cell transfer should consist of a heterogeneous mixture of memory cells.

Hybrid polypeptide heavy chains produced by two hybridoma lines.

Two anti-TNP antibodies exhibiting unusual features are described. They were obtained in two independent fusions. Spleen cells from CB20 mice sensitized with TNP-Ficoll and challenged with TNP-LPS were fused with SP2/0 myeloma cells. One of these hybridomas, CBT3, secretes antibodies which react with both monospecific anti-gamma 2b and anti-gamma 3 anti-isotypic sera; the second hybridoma, CBT4, secretes antibodies reacting with monospecific anti-mu and anti-gamma 2b sera. Only one type of immunoglobulin is secreted by each hybridoma, ruling out the hypothesis of hybrid molecules formed by distinct heavy chains. These results imply that the two heavy chains are made up from elements encoded by gamma 3 and gamma 2b genes in CBT3 and by gamma 2b and mu genes in CBT4. The molecular mechanisms underlying the production of these singular heavy chains are discussed.

A description of two morphologic patterns of aortic fatty streaks, and a hypothesis of their pathogenesis.

Two morphologic patterns of fatty streak were identified on examination of 74 aortas from the Pathobiological Determinants of Atherosclerosis in Youth study. Pattern 1, which predominated in 78% of aortas, is characterized by broad bands of intense stain which extend to the proximal edge of ostia. Pattern 2, which predominated in 11%, is characterized by less intense staining which is concave to the associated ostium. Pattern 1 predominated in older subjects and smokers. Aging and smoking decrease arterial elasticity, thereby decreasing the volume and duration of retrograde blood flow in diastole. Doppler ultrasonography of the posterior intercostal arteries and aorta in 42 healthy subjects revealed that retrograde blood flow in late systole/early diastole is normal in subjects in the 15-34 age group. Transition from retrograde to antegrade flow was associated with transient blood stasis. This stasis should prolong the residence time of lipid-rich particles, enhancing diffusion into the vessel wall. A region of lower flow velocity was noted in the periostial region in all patients during diastole. The anatomic, hemodynamic, and risk factor data suggest that the morphology of fatty streaks is determined by interaction of retrograde with antegrade blood flow as modulated by arterial elasticity.

Increased peak blood velocity in association with elevated blood pressure.

To test the hypothesis that peak blood velocity in the common carotid artery is increased in association with elevated blood pressure, the authors measured peak common carotid blood velocity in 458 subjects by color Doppler ultrasonography. Blood pressure was measured at the time of ultrasound examination by automated sphygmomanometer. Peak blood velocity was increased in subjects with elevated blood pressure (right common carotid: 72.5 +/- 2.0 cm/s vs. 62.7 +/- 2.5 cm/s, left common carotid: 72.0 +/- 1.8 cm/s vs. 63.9 +/- 2.0 cm/s, p < 0.001). Peak blood velocity was significantly correlated with systolic blood pressures between 135 and 160 mmHg (r = 0.47 in right common carotid, 0.45 in left common carotid, n = 123, p < 0.001). No correlation was found between peak blood velocity and blood pressures less than 135 mmHg or greater than 160 mmHg. By increasing erythrocyte momentum, increased peak blood velocity may play a role in the pathogenesis of arterial diseases associated with hypertension.

Common bile duct measurements in an elderly population.

We prospectively evaluated the diameter of the common bile duct in 1,018 patients between the ages of 60 to 96 over a 4 year period to determine if there is a significant change in its size with aging. All of the patients included in the study were being evaluated primarily for carotid or peripheral vascular disease. Any patients with a history of biliary disease (i.e., bilirubin level greater than 1.5 mg/ml, cholecystectomy, or cholelithiasis) were excluded. Ultrasonography of the common bile duct was performed only in those patients with no subjective abdominal pain or icterus. Our results demonstrated a small although statistically significant increase in the caliber of the common bile duct with increasing age (60 years old or less, mean diameter 3.6 mm +/- 0.2mm, versus over 85 years old, mean diameter 4 mm +/- 0.2 mm, P = 0.009). Although the common bile duct did increase in size with aging, 98% of all ducts remained below 6 to 7 mm, the commonly accepted upper range of normal.

Opposite effects of interleukin-4 on memory T helper cell development depend on interleukin-2.

We previously reported that cyclosporin A (CSA) promotes the generation of T helper memory cells during antigenic priming of murine spleen cells in vitro. More recently, we have demonstrated that interleukin-2 (IL2) has a downmodulating effect on T helper memory cell generation. The present data address the role of the other T cell growth factor, IL4, upon induction of these cells. The data presented here show that IL4 can interfere with this process: addition of rIL4 to immunosuppressed priming cultures leads to a considerable decrease in the helper activity of the recovered cells. However, in standard cultures, in which IL2 is normally produced, no effect of IL4 on T helper memory cell generation was found. Addition of IL4 has important consequences for cytokines produced upon antigenic restimulation. In standard cultures, IL4 primes for cells expressing high levels of IL2 and IL4 mRNA. Strikingly, in immunosuppressed priming cultures, IL4 counterbalances the CSA-induced blockade of the IFN gamma gene. Taken together, our results suggest that the unique role of IL4 is to drive T helper memory precursors into an IL4 production differentiation pathway. However, IL4 has a downmodulating effect on memory T helper cell induction when IL2 is not produced. These results confirm that synergy between IL2 and IL4 is mandatory for the directive role of IL4 upon IL4-producing cells. Furthermore, the finding that IL4 promotes the induction of IFN gamma in a CSA-resistant pathway represents a new tool for analysis of regulation of the IFN gamma gene.

Interleukin-2 down-modulates memory T helper lymphocyte development during antigenic stimulation in vitro.

Using an in vitro antigenic stimulation model of murine spleen cells in the presence of the immunosuppressor cyclosporin A (CSA) we have previously reported that not only does this drug not interfere with the differentiation of T lymphocytes into memory cells it appears to favor this differentiation (Motta, I. et al., Eur. J. Immunol. 1991. 21:551). Because CSA blocks interleukin-2 (IL-2) gene expression, we have analyzed the effect of this cytokine on memory T helper cell development. Murine splenic cells were primed for 6 days with sheep red blood cells (SRBC) in protocols in which either IL-2 was not produced or its biological activity was neutralized by anti-IL-2 receptor (R) antibodies. The helper function of the recovered T cells was revealed by their capacity to help virgin B splenocytes produce anti-SRBC antibodies upon challenge in vitro. We found that CD4+ cells primed in the absence of IL-2, provoked either by IL-2 gene transcription blockade by CSA or by treatment with anti-IL-2R antibodies, afford the best helper functions. These cells exhibit a memory-type phenotype characterized by the low expression of the MEL-14 marker and the high expression of the CD44 marker. Evidence is also presented that memory T helper cells originate in part from naive subset displaying the MEL-14hi phenotype. The pattern of expression of the genes encoding different cytokines (IL-2, IL-4, IL-5 and interferon-gamma) following a secondary antigenic stimulation shows that the helper function of the cells primed in the absence of IL-2 correlates with the up-regulation of the IL-2 and the IL-5 genes. From these data, we conclude that IL-2 plays a major role in the control of memory T helper cell induction.

[Diagnosis of hydroxyapatite disease]. / Beitrag zur Diagnostik der Hydroxyapatitkrankheit.

The possibility of detecting hydroxyapatite crystals in synovia in a routine setting has been studied prospectively. Coloration of the crystals with alizarin red-S was the method of choice. The diagnostic results were markedly improved by simultaneous observation of a freshly prepared positive control synovia.

The expression of the sIgD isotype in wild-derived mice.

IgD and IgM are concomitantly expressed on the surface of most mouse B lymphocytes and both molecules serve as receptor for antigen. In this communication we report that in contrast to IgM, which is expressed in a constant manner on the surface of spleen B lymphocytes of different laboratory and wild-derived mice, IgD expression is variable among the spleen cells of wild-derived mice. SPE, SEI, and SFM mice belonging to the Mus 3 subgroup show a fluorescence profile characterized by a marked diminution in the population of B lymphocytes expressing the IgD isotype; in addition, these cells have a low sIgD density on their membranes. These findings were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the iodinated membrane proteins. Polyclonal in vitro activation with lipopolysaccharide increases the frequency of surface IgD (sIgD)-bearing spleen cells and sIgD density in the SPE strain but decreases both the frequency and the density of IgD bearing cells in the BALB/c strain. This result suggests that delta gene expression is regulated differently in SPE and BALB/c mice. In addition, genetic analysis of sIgD expression in (BALB/c X SPE)F1 hybrids suggests that the proportion of sIgD-bearing cells and sIgD density are independently regulated.

Colchicum autumnale agglutinin activates all murine T-lymphocytes but does not induce the proliferation of all activated cells.

Plant lectins with mitogenic properties for T-lymphocytes have been particularly useful for the study of T-cell activation and effector functions. In the search for mitogenic lectins possessing activation features different from the ones associated with the already known mitogens, we found that an agglutinin isolated from Colchicum autumnale tubers, Colchicum autumnale agglutinin (CAA), possesses interesting properties. First, contrasting with the classical mitogens, CAA induces the proliferation of a fraction of the CD4+ and CD8+ mouse T-lymphocytes. Second, the CAA-induced proliferation requires MHC class II and CD4 molecules. Third, although only a fraction of T-cells enters into the cell cycle, all T-lymphocytes are activated and express high levels of the activation markers CD69 and CD44. Finally, CAA-stimulation is characterized by a particular pattern of the cytokine gene expression, reflected by the transcription of the IL2, IL5, and IFN-gamma genes, while the IL4 and IL10 genes remained silent. Taken together these data demonstrate that CAA activation does not conform to the pathway of T-cell triggering observed with classical mitogenes and represents a new tool for the analysis of T-cell activation.

Cytogenetic study in spermatocytes of mice and Chinese hamsters after treatment with isoniazid (INH).

Meiotic chromosomes of spermatocytes from INH-treated male mice and Chinese hamsters were analysed for chromosome aberrations in diakinesis-metaphase I and metaphase II. The experiments were performed in two laboratories while a third laboratory participated in the chromosome evaluation. No enhancement of chromosome aberrations could be observed after acute treatment of early primary spermatocytes or chronic treatment of spermatogonia with INH.

Differences in helper T-cell sensitivity to anti-thy-1.2 alloantiserum or monoclonal antibody.

A Thy-1 alloantigen recognized by a monoclonal antibody which is not present in all strains of mice carrying the Thy-1b allele is reported. Indeed, helper and memory T cells of DBA/2 strain are not eliminated by the monoclonal antibody used in this study.

A new approach to immunological memory using thymus-independent antigens.

Contrary to what has been reported of thymus-independent antigens, we recently demonstrated that trinitrophenylated lipopolysaccharide (TNP-LPS), a class 1 thymus-independent antigen, elicited an anti-TNP anamnestic response in C57BL/6 mice. The question of whether or not class 2 thymus-independent antigens (DNP-Ficoll and DNP-dextran) could also induce immunological memory in this mouse strain was examined. Evidence induce immunological memory in this mouse strain was examined. Evidence is presented that priming with either of these class 2 thymus-independent antigens resulted in the induction of memory B lymphocytes. However, while the memory cells generated by these two antigens were able to be activated by TNP-LPS, they were not triggered by class 2 thymus-independent antigens. Genetic analysis of the capacity of different mouse strains to mount a secondary response to TNP-LPS revealed that major histo-compatibility-associated genes did not play an essential role, but that IgH-V or closely linked gene(s) controlled the immunological memory to TNP-LPS. These findings are discussed in terms of regulatory phenomena which govern the expression of memory response to thymus-independent antigens.

Hepatic helical CT: contrast material injection protocol.

PURPOSE: To develop and compare contrast material injection protocols suitable for hepatic helical computed tomography (CT). MATERIALS AND METHODS: Monophasic and biphasic helical CT were performed with contrast material with an iodine load of 50 g at 3 mL/sec for 60 seconds or at 5 mL/sec for 10 seconds and 2 mL/sec for 65 seconds, respectively. In 58 men and 51 women, aged 22-77 years, aortic and hepatic enhancement curves were constructed from a cluster acquisition with a slip-ring scanner operating in a nonhelical mode. RESULTS: The monophasic protocol produced a higher peak aortic enhancement (180 HU +/- 47 [+/- 1 standard deviation]) than the biphasic protocol (150 HU +/- 24). Peak hepatic enhancement (63-64 HU +/- 15) was equivalent. Calculated equilibrium time for the monophasic protocol was 95.1 seconds and for the biphasic protocol was 101.4 seconds. The contrast enhancement index differed only marginally between the two protocols (P < .4). CONCLUSION: Monophasic and biphasic protocols produced equivalent results when tailored for the shorter temporal window of a rapid-sequence helical acquisition.

Is carbohydrate-deficient transferrin a specific marker for alcohol abuse? A study in patients with chronic viral hepatitis.

Carbohydrate-deficient transferrin, a transferrin isoform, is hailed as a new marker of chronic alcohol abuse, but its specificity is, however, not unequivocally accepted. The aim of the present study was therefore to determine carbohydrate-deficient transferrin levels in patients with chronic hepatitis B and C with or without documented chronic alcohol intake. Carbohydrate-deficient transferrin was measured using a double-antibody radioimmunoassay (CDTect, Pharmacia) in serum samples from 66 patients (45 males and 21 females; mean age: 39 years) with chronic viral hepatitis B (n = 20) or C (n = 46). Diagnosis of the underlying liver disease was established by liver biopsy. Carbohydrate-deficient transferrin levels were raised in 15 patients [23%; hepatitis B (n = 2) and hepatitis C (n = 13)]. In patients with chronic hepatitis B, the carbohydrate-deficient transferrin level was raised in two abstainers. In the 46 patients with chronic hepatitis C, 10 (22%) patients with an alcohol consumption of < 60 g/day for the men and 30 g/day for the women had raised carbohydrate-deficient transferrin levels. The overall specificity of carbohydrate-deficient transferrin for chronic alcohol abuse was thus 78%, suggesting an association between elevated carbohydrate-deficient transferrin levels and the presence of chronic viral hepatitis. Carbohydrate-deficient transferrin levels were not correlated with the histological grading or staging of chronic hepatitis B and C, or with biological markers of hepatic synthesis and cellular damage. Thus, an increased carbohydrate-deficient transferrin level may occur in patients with chronic viral hepatitis in the absence of chronic alcohol abuse. This fact should be kept in mind by physicians when using this marker to detect alcohol abuse.

Periportal halo: a CT sign of liver disease.

Periportal halos are defined as circumferential zones of decreased attenuation identified around the peripheral or subsegmental portal venous branches on contrast-enhanced computed tomography (CT). These halos probably represent fluid or dilated lymphatics in the loose areolar zone around the portal triad structures. While this CT finding is nonspecific, it is abnormal and should prompt close scrutiny of the liver in search of an underlying etiology. Periportal halos which may be due to blood are commonly seen in patients with liver trauma. Periportal edema may cause this sign in patients with congestive heart failure and secondary liver congesion, hepatitis, or enlarged lymph nodes and tumors in the porta hepatis which obstruct lymphatic drainage. This CT sign has also been observed in liver transplants (probably secondary to disruption and engorgement of lymphatic channels) and in recipients of bone marrow transplants who might develop liver edema from microvenous occlusive disease. While the precise pathophysiologic basis of periportal tracking has not been proven, it represents a potentially important CT sign of occult liver disease.

Diagnosis of ectopic pregnancy: endovaginal vs transabdominal sonography.

During a 25-month period, 193 women with the clinical diagnosis of suspected ectopic pregnancy had transabdominal and endovaginal sonograms. Most had quantitative determinations of serum human chorionic gonadotropin (HCG). Endovaginal sonography was diagnostic of ectopic pregnancy in 23 (38%) of the 60 patients with surgically proved ectopic pregnancies: transabdominal sonography was diagnostic in 13 patients (22%). All 83 intrauterine pregnancies were identified with endovaginal sonography, compared with 34 identified with transabdominal sonography. Endovaginal sonography was somewhat more helpful in the diagnosis of missed abortion and blighted ovum. Eighty endovaginal sonograms were classified as indeterminate as compared with 141 transabdominal studies. This indeterminate group included patients with complete abortions, ectopic pregnancies without sonographic evidence of an extrauterine gestation, incomplete abortions, and patients with subsequent negative serum levels. As in prior reports, endovaginal sonography was superior to transabdominal sonography in the evaluation of suspected ectopic pregnancies. Overall, endovaginal sonography was diagnostic in 113 patients, whereas transabdominal sonography was diagnostic in 52 patients. The finding of an extrauterine fetal pole or embryo was diagnostic for an ectopic pregnancy. Pelvic fluid, the appearance of the endometrium, and a single positive serum HCG determination were not helpful in making the diagnosis of ectopic pregnancy.