Novel inhibitors that target bacterial virulence identified via HTS against intra-macrophage survival of Shigella flexneri.

Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a "pre-antibiotic" type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.

Stress-triggered hematopoietic stem cell proliferation relies on PrimPol-mediated repriming.

Stem cell division is linked to tumorigenesis by yet-elusive mechanisms. The hematopoietic system reacts to stress by triggering hematopoietic stem and progenitor cell (HSPC) proliferation, which can be accompanied by chromosomal breakage in activated hematopoietic stem cells (HSCs). However, whether these lesions persist in their downstream progeny and induce a canonical DNA damage response (DDR) remains unclear. Inducing HSPC proliferation by simulated viral infection, we report that the associated DNA damage is restricted to HSCs and that proliferating HSCs rewire their DDR upon endogenous and clastogen-induced damage. Combining transcriptomics, single-cell and single-molecule assays on murine bone marrow cells, we found accelerated fork progression in stimulated HSPCs, reflecting engagement of PrimPol-dependent repriming, at the expense of replication fork reversal. Ultimately, competitive bone marrow transplantation revealed the requirement of PrimPol for efficient HSC amplification and bone marrow reconstitution. Hence, fine-tuning replication fork plasticity is essential to support stem cell functionality upon proliferation stimuli.

Morphological Description of the Early Events during the Invasion of Acanthamoeba castellanii Trophozoites in a Murine Model of Skin Irradiated under UV-B Light.

Skin infections have been associated with Acanthamoeba, nevertheless the events during skin invasion and UV-B light effects on it are unknown. The early morphological events of Acanthamoeba castellanii skin invasion are shown in SKH-1 mice that were chronically UV-B light irradiated. Mice that developed skin lesions (group 1) were topical and intradermally inoculated with A. castellanii trophozoites and sacrificed 48 h or 18 days later. Mice that showed no skin lesions (group 2) were intradermally inoculated and sacrificed 24, 48 or 72 h later. Mice ventral areas were considered controls with and without trophozoites intradermally inoculated. Skin samples were processed by histological and immunohistochemistry techniques. In group 1, trophozoites were immunolocalized in dermal areas, hair cysts, sebaceous glands, and blood vessels, and collagen degradation was observed. One of these mice shown trophozoites in the spleen, liver, and brain. In group 2, few trophozoites nearby collagenolytic activity zones were observed. In control samples, nor histological damage and no trophozoites were observed. Adherence and collagenolytic activity by A. castellanii were corroborated in vitro. We can infer that UV-B light irradiated skin could favor A. castellanii invasiveness causing damage in sites as far away as the brain, confirming the invasive capacity and pathogenic potential of these amphizoic amoebae.

Schwann Cell Autophagy and Necrosis as Mechanisms of Cell Death by Acanthamoeba.

Amoebae of the genus Acanthamoeba are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, Acanthamoeba trophozoites were immunolocalized in contact with the peripheral nervous system (PNS) cells-Schwann cells (SC). In this study, we analyzed in greater detail the in vitro early morphological events (1, 2, 3, and 4 h) during the interaction of A. culbertsoni trophozoites (ATCC 30171) with SC from Rattus norvegicus (ATCC CRL-2941). Samples were processed for scanning and transmission electron microscopy as well as confocal microscopy. After 1 h of interaction, amoebae were observed to be adhered to the SC cultures, emitting sucker-like structures associated with micro-phagocytic channels. In addition, evidence of necrosis was identified since edematous organelles as well as multivesicular and multilamellar bodies characteristics of autophagy were detected. At 2 h, trophozoites migrated beneath the SC culture in which necrosis and autophagy persisted. By 3 and 4 h, extensive lytic zones were observed. SC necrosis was confirmed by confocal microscopy. We reported for the first time the induction of autophagic and necrotic processes in PNS cells, associated in part with the contact-dependent pathogenic mechanisms of A. culbertsoni trophozoites.

Genetic, Immunological, and Clinical Features of the First Mexican Cohort of Patients with Chronic Granulomatous Disease.

PURPOSE: Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by an inability of phagocytes to produce reactive oxygen species, impairing their killing of various bacteria and fungi. We summarize here the 93 cases of CGD diagnosed in Mexico from 2011 to 2019. METHODS: Thirteen Mexican hospitals participated in this study. We describe the genetic, immunological, and clinical features of the 93 CGD patients from 78 unrelated kindreds. RESULTS: Eighty-two of the patients (88%) were male. All patients developed bacterial infections and 30% suffered from some kind of fungal infection. Fifty-four BCG-vaccinated patients (58%) presented infectious complications of BCG vaccine. Tuberculosis occurred in 29%. Granulomas were found in 56% of the patients. Autoimmune and inflammatory diseases were present in 15% of patients. A biological diagnosis of CGD was made in 89/93 patients, on the basis of NBT assay (n = 6), DHR (n = 27), and NBT plus DHR (n = 56). The deficiency was complete in all patients. The median age of biological diagnosis was 17 months (range, 0-186 months). A genetic diagnosis was made in 83/93 patients (when material was available), corresponding to CYBB (n = 64), NCF1 (n = 7), NCF2 (n = 7), and CYBA (n = 5) mutations. CONCLUSIONS: The clinical manifestations in these Mexican CGD patients were similar to those in patients elsewhere. This cohort is the largest in Latin America. Mycobacterial infections are an important cause of morbidity in Mexico, as in other countries in which tuberculosis is endemic and infants are vaccinated with BCG. X-linked CGD accounted for most of the cases in Mexico, as in other Latin American countries. However, a significant number of CYBA and NCF2 mutations were identified, expanding the spectrum of known causal mutations.

Interobserver variability in upfront dichotomous histopathological assessment of ductal carcinoma in situ of the breast: the DCISion study.

Histopathological assessment of ductal carcinoma in situ, a nonobligate precursor of invasive breast cancer, is characterized by considerable interobserver variability. Previously, post hoc dichotomization of multicategorical variables was used to determine the "ideal" cutoffs for dichotomous assessment. The present international multicenter study evaluated interobserver variability among 39 pathologists who performed upfront dichotomous evaluation of 149 consecutive ductal carcinomas in situ. All pathologists independently assessed nuclear atypia, necrosis, solid ductal carcinoma in situ architecture, calcifications, stromal architecture, and lobular cancerization in one digital slide per lesion. Stromal inflammation was assessed semiquantitatively. Tumor-infiltrating lymphocytes were quantified as percentages and dichotomously assessed with a cutoff at 50%. Krippendorff's alpha (KA), Cohen's kappa and intraclass correlation coefficient were calculated for the appropriate variables. Lobular cancerization (KA = 0.396), nuclear atypia (KA = 0.422), and stromal architecture (KA = 0.450) showed the highest interobserver variability. Stromal inflammation (KA = 0.564), dichotomously assessed tumor-infiltrating lymphocytes (KA = 0.520), and comedonecrosis (KA = 0.539) showed slightly lower interobserver disagreement. Solid ductal carcinoma in situ architecture (KA = 0.602) and calcifications (KA = 0.676) presented with the lowest interobserver variability. Semiquantitative assessment of stromal inflammation resulted in a slightly higher interobserver concordance than upfront dichotomous tumor-infiltrating lymphocytes assessment (KA = 0.564 versus KA = 0.520). High stromal inflammation corresponded best with dichotomously assessed tumor-infiltrating lymphocytes when the cutoff was set at 10% (kappa = 0.881). Nevertheless, a post hoc tumor-infiltrating lymphocytes cutoff set at 20% resulted in the highest interobserver agreement (KA = 0.669). Despite upfront dichotomous evaluation, the interobserver variability remains considerable and is at most acceptable, although it varies among the different histopathological features. Future studies should investigate its impact on ductal carcinoma in situ prognostication. Forthcoming machine learning algorithms may be useful to tackle this substantial diagnostic challenge.

Evaluation of the sensitivity to chlorhexidine, voriconazole and itraconazole of T4 genotype Acanthamoeba isolated from Mexico.

Free-living amoebae of the genus Acanthamoeba are the etiological agents of cutaneous lesions, granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK), which are chronic infections with poor prognosis if not diagnosed promptly. Currently, there is no optimal therapeutic scheme to eradicate the pathologies these protozoa cause. In this study we report the morphological and molecular identification of three species of the genus Acanthamoeba, belonging to T4 group; A. polyphaga isolated from the corneal ulcer of a patient sample of AK case; A. castellanii isolated from the contact lens of an AK patient and A. palestinensis obtained from a soil sample. The in vitro activity of chlorhexidine, itraconazole and voriconazole drugs against trophic stage was also evaluated through a colorimetric assay based on the oxidation-reduction of alamar blue. The strains in the study were sensitive to the evaluated drugs; although when determining the 50% inhibitory concentration (IC50) statistically significant differences were observed. A. castellanii showed to be highly sensitive to voriconazole (0.66 ±â€¯0.13 µM) but the least sensitive to chlorhexidine and itraconazole (8.61 ±â€¯1.63 and 20.14 ±â€¯4.93 µM, respectively), A. palestinensis showed the highest sensitivity to itraconazole (0.502 ±â€¯0.11 µM) and A. polyphaga expressed moderate sensitivity to chlorhexidine and itraconazole and lower sensitivity to voriconazole (10.10 ±â€¯2.21 µM). These results showed that species of the genus Acanthamoeba express different sensitivity to the tested drugs, which could explain the problems surrounding the establishment of a treatment of choice in the infections caused by these amoebae. We consider that although chlorhexidine and itraconazole show good activity on these amoebae and have been used in cases of AK in Mexico with acceptable results, voriconazole should be considered as the first therapeutic option of future Acanthamoeba infections that will be diagnosed in our country.

Acanthamoeba keratitis in Mexico: Report of a clinical case and importance of sensitivity assays for a better outcome.

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection. The early symptoms include redness, pain, photophobia and intense tearing. Chronic infection usually progresses to stromal inflammation, ring ulcers, corneal opacification and hypopyon. Here we document an AK case in a high myopic 38-year-old woman from Mexico City, with a history of wearing contact lenses while swimming. Corneal scrapes cultures were positive only for amoebae, consequently a treatment including netilmicin 0.3% and oral itraconazole 100 mg/12 h was prescribed. The infection was resolved after 8 months, leaving a slight leucoma outside the visual axis, with a visual acuity of 20/150. In the laboratory, the amoebic isolate was axenized in PYG medium, with an optimal growth at 30 °C, and was identified morphologically as Acanthamoeba polyphaga according to the taxonomic criteria of Page (1988) and placed in the T4 group by genotyping. The virulence of this strain (40%) was determined by intranasal inoculation of 1 × 106/20 µl trophozoites in BALB/c mice recovering from brain, proving their invasion ability and by the interaction with monolayers of epithelial cells of the established MDCK line of canine kidney origin (1:2 ratio of interaction), at 1, 3, 6, 8 and 24 h; trophozoites migrated to cell junctions inducing few lytic zones. In addition to the biological characterization, in vitro drug sensitivity tests were performed using chlorhexidine, itraconazole, netilmicin and voriconazole. Results revealed that voriconazole was the most effective compound. A. polyphaga remains as one of the most frequently isolated species producing AK. The treatment of AK case using netilmicin and oral itraconazole solved the disease, but the healing process was wide-ranging (8 months). The use of voriconazole and chlorhexidine may be an alternative treatment of future AK cases in Mexico.

Diagnóstico y tratamiento histeroscópico de una paciente posmenopáusica con fístula arteriovenosa uterina / Hysteroscopy diagnosis and management of uterine arterio-venous fistula in a postmenopausal woman

Resumen ANTECEDENTES: La fístula arteriovenosa uterina es una de las malformaciones vasculares más frecuentes. El sangrado vaginal abundante es un signo sugerente de este tipo de alteración. CASO CLÍNICO: Paciente de 51 años, acudió al servicio de Urgencias debido a un sangrado vaginal abundante de un mes de evolución. En la exploración física se objetivaron los genitales externos y la vagina normales, y el útero ligeramente aumentado de tamaño. La ecografía transvaginal con Doppler color solo reportó aumento de la vascularización uterina. La histeroscopia diagnóstica confirmó la coexistencia de una fístula arteriovenosa uterina. La histeroscopia quirúrgica se practicó con espéculo, pinza de Pozzi, dilatación con tallos de Hegar (9'5), coagulación con histeroscopio Storz, asa de Collins, con energía bipolar a 45 watt y suero fisiológico como medio de distensión. La malformación arteriovenosa se coaguló en diversas zonas anatómicas, hasta conseguir la obstrucción completa de la fístula. El posoperatorio trascurrió sin contratiempos. El seguimiento de la paciente fue satisfactorio, hasta la fecha no manifiesta síntomas adicionales de la alteración. CONCLUSIONES: La histeroscopia es un método efectivo, de mínima invasión y con escasa morbilidad para establecer el diagnóstico y tratamiento de las malformaciones vasculares uterinas.

Abstract BACKGROUND: Uterine arterio-venous fistula is one of the most frequent vascular malformations. A heavy vaginal bleeding should make us suspect the presence of this type of vascular malformation. CLINICAL CASE: A 51-years-old patient was admitted in Emergency Service for heavy vaginal bleeding of one month of evolution. On physical examination, external genitalia and vagina were normal but uterus had slightly increased in size. A transvaginal ultrasound with color Doppler was performed where an increase in uterine vascularization was shown. Subsequently, an office hysteroscopy was carried out confirming the diagnosis of uterine arterio-venous fistula. After a correct anesthetic evaluation, a surgical hysteroscopy was performed, using speculum, Pozzi clamp, dilation with Hegar stems up to number 9.5, coagulation resectoscope Storz Gyrus with Collins loop handle with bipolar energy at 45 watt of coagulation and physiological saline, as a means of relaxation. The arterio-venous malformation was coagulated at several levels until the obliteration of the fistula was completed. The postoperative period was favorable. In the subsequent follow-up in consultation, the patient continues asymptomatic. CONCLUSION: Hysteroscopy is an effective, minimally invasive method with low morbidity to establish the diagnosis and treatment of vascular malformations.

Functional interplay between c-Myc and Max in B lymphocyte differentiation.

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix-loop-helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c-Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c-Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c-Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c-Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B-cell receptor. These data suggest that c-Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine-tuning the initial response after activation.

Type 2 diabetes mellitus BALB/c mice are more susceptible to granulomatous amoebic encephalitis: Immunohistochemical study.

Granulomatous amoebic encephalitis (GAE) is a chronic, difficult to resolve infection caused by amphizoic amoebae of the genus Acanthamoeba, which in most cases occurs in immunosuppressed persons or with chronic diseases such as diabetes. In this study, we describe the early events of A. culbertsoni infection of GAE in diabetic mice model. Diabetes was induced in male BALB/c mice, with a dose of streptozotocin (130 mg/kg). Healthy and diabetic mice were inoculated via intranasal with 1 × 106 trophozoites of A. culbertsoni. Then were sacrificed and fixed by perfusion at 24, 48, 72 and 96 h post-inoculation, the brains and nasopharyngeal meatus were processed to immunohistochemical analysis. Invasion of trophozoites in diabetic mice was significantly greater with respect to inoculated healthy mice. Trophozoites and scarce cysts were immunolocalized in respiratory epithelial adjacent bone tissue, olfactory nerve packets, Schwann cells and the epineurium base since early 24 h post-inoculation. After 48 h, trophozoites were observed in the respiratory epithelium, white matter of the brain, subcortical central cortex and nasopharyngeal associated lymphoid tissue (NALT). At 72 h, cysts and trophozoites were immunolocalized in the olfactory bulb with the presence of a low inflammatory infiltrate characterized by polymorphonuclear cells. Scarce amoebae were observed in the granular layer of the cerebellum without evidence of inflammation or tissue damage. No amoebas were observed at 96 h after inoculation, suggesting penetration to other tissues at this time. In line with this, no inflammatory infiltrate was observed in the surrounding tissues where the amoebae were immunolocalized, which could contribute to the rapid spread of infection, particularly in diabetic mice. All data suggest that trophozoites invade the tissues by separating the superficial cells, penetrating between the junctions without causing cytolytic effect in the adjacent cells and subsequently reaching the CNS, importantly, diabetes increases the susceptibility to amoebae infection, which could favor the GAE development.

p21Cip1 plays a critical role in the physiological adaptation to fasting through activation of PPARα.

Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the role of stress-responsive tumor suppressors in fasting. Here, we have examined the expression of several tumor suppressors upon fasting in mice. Interestingly, p21 mRNA is uniquely induced in all the tissues tested, particularly in liver and muscle (>10 fold), and this upregulation is independent of p53. Remarkably, in contrast to wild-type mice, p21-null mice become severely morbid after prolonged fasting. The defective adaptation to fasting of p21-null mice is associated to elevated energy expenditure, accelerated depletion of fat stores, and premature activation of protein catabolism in the muscle. Analysis of the liver transcriptome and cell-based assays revealed that the absence of p21 partially impairs the transcriptional program of PPARα, a key regulator of fasting metabolism. Finally, treatment of p21-null mice with a PPARα agonist substantially protects them from their accelerated loss of fat upon fasting. We conclude that p21 plays a relevant role in fasting adaptation through the positive regulation of PPARα.

Partial Loss of Rpl11 in Adult Mice Recapitulates Diamond-Blackfan Anemia and Promotes Lymphomagenesis.

Diamond-Blackfan anemia (DBA) is characterized by anemia and cancer susceptibility and is caused by mutations in ribosomal genes, including RPL11. Here, we report that Rpl11-heterozygous mouse embryos are not viable and that Rpl11 homozygous deletion in adult mice results in death within a few weeks, accompanied by bone marrow aplasia and intestinal atrophy. Importantly, Rpl11 heterozygous deletion in adult mice results in anemia associated with decreased erythroid progenitors and defective erythroid maturation. These defects are also present in mice transplanted with inducible heterozygous Rpl11 bone marrow and, therefore, are intrinsic to the hematopoietic system. Additionally, heterozygous Rpl11 mice present increased susceptibility to radiation-induced lymphomagenesis. In this regard, total or partial deletion of Rpl11 compromises p53 activation upon ribosomal stress or DNA damage in fibroblasts. Moreover, fibroblasts and hematopoietic tissues from heterozygous Rpl11 mice present higher basal cMYC levels. We conclude that Rpl11-deficient mice recapitulate DBA disorder, including cancer predisposition.

Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality.

Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These 'dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality.

Subclavicular pectoralis major myocutaneous flap for optimal reconstruction of large orbitozygomatic defects: a case report.

Since the introduction of microvascular free flaps, the pectoralis major myocutaneous flap (PMMF) has been relegated to background for most reconstructive surgeons. The objective of this article is to show the advantages of cervicofacial defects reconstruction with PMMF using the subclavicular plane route in a challenging clinical case. An 83-year-old man presented with cutaneous temporomalar lesion with orbital spread. Tumor resection was performed, including 12 × 11 cm skin and subcutaneous tissue, overlying zygomatic and malar bone, and orbital exenteration. Radical parotidectomy and functional neck dissection were performed. PMMF was chosen as reconstructive option routing the pedicle to the subclavicular plane. The length of the pedicle was 31 cm. The subclavicular route for PMMF increases the flap's length and arc of rotation compared with the conventional supraclavicular one. This procedure decreases the bulk of the PMMF pedicle which makes it functionally and cosmetically favorable. By using this modification, we may widen the "safe" reconstructive possibilities.

Cdk4 and Cdk6 cooperate in counteracting the INK4 family of inhibitors during murine leukemogenesis.

Cdk4 and Cdk6 are related protein kinases that bind d-type cyclins and regulate cell-cycle progression. Cdk4/6 inhibitors are currently being used in advanced clinical trials and show great promise against many types of tumors. Cdk4 and Cdk6 are inhibited by INK4 proteins, which exert tumor-suppressing functions. To test the significance of this inhibitory mechanism, we generated knock-in mice that express a Cdk6 mutant (Cdk6 R31C) insensitive to INK4-mediated inhibition. Cdk6(R/R) mice display altered development of the hematopoietic system without enhanced tumor susceptibility, either in the presence or absence of p53. Unexpectedly, Cdk6 R31C impairs the potential of hematopoietic progenitors to repopulate upon adoptive transfer or after 5-fluorouracil-induced damage. The defects are overcome by eliminating sensitivity of cells to INK4 inhibitors by introducing the INK4-insensitive Cdk4 R24C allele, and INK4-resistant mice are more susceptible to hematopoietic and endocrine tumors. In BCR-ABL-transformed hematopoietic cells, Cdk6 R31C causes increased binding of p16(INK4a) to wild-type Cdk4, whereas cells harboring Cdk4 R24C and Cdk6 R31C are fully insensitive to INK4 inhibitors, resulting in accelerated disease onset. Our observations reveal that Cdk4 and Cdk6 cooperate in hematopoietic tumor development and suggest a role for Cdk6 in sequestering INK4 proteins away from Cdk4.

Reprogramming in vivo produces teratomas and iPS cells with totipotency features.

Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

The proto-oncogene c-myc regulates antibody secretion and Ig class switch recombination.

The immune response involves the generation of Ab-secreting cells and memory B cells through a process called terminal B lymphocyte differentiation. This program requires the transcriptional repressor Blimp-1, which inhibits c-myc expression and terminates proliferation. Although the role of c-Myc in cell proliferation is well characterized, it is not known whether it has other functions in terminal differentiation. In this study, we show that c-Myc not only regulates cell proliferation, but it is also essential for Ab-secreting cell function and differentiation in vivo. c-Myc-deficient B lymphocytes hypersecrete IgM and do not undergo Ig class switch recombination (CSR). CSR has been previously linked to proliferation, and in this study we mechanistically link class switching and proliferation via c-Myc. We observed that c-Myc regulates CSR by transcriptionally activating the B cell-specific factor activation-induced cytidine deaminase. By linking cell proliferation and CSR, c-Myc is thus a critical component for a potent immune response.

Detection of genomic abnormalities in multiple myeloma: the application of FISH analysis in combination with various plasma cell enrichment techniques.

Multiple myeloma (MM) is a hematopoietic neoplasm characterized by malignant plasma cells (PCs) that accumulate in the bone marrow. A number of different genomic abnormalities are associated with MM; however, detection of these by fluorescence in situ hybridization (FISH) can be limited by the percentage of PCs in the specimen. In this study, we tested 20 bone marrow specimens with known MM and a low concentration of monoclonal PCs for the presence of genomic abnormalities using FISH in combination with various PC enrichment techniques: magnetic cell sorting, targeted manual scoring, and automated image analysis. In addition, flow cytometric cell sorting of PCs in combination with FISH analysis was also tested for minimal residual disease applications. Different parameters were evaluated when assessing the detection efficiency of each approach. FISH results are highly dependent on the chosen enrichment method. We describe the evaluation of different techniques applicable for various laboratory settings and specimen parameters.