Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers.

BACKGROUND: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. METHODS: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. RESULTS: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. CONCLUSION: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.

[Management of rhegmatogenous retinal detachment with unseen breaks: about 50 cases]. / Prise en charge des decollements de retine rhegmatogenes sans dechirure visible: a propos de 50 cas.

PURPOSE: To evaluate the clinical and therapeutic characteristics of rhegmatogenous retinal detachment (RRD) with unseen retinal breaks. PATIENTS AND METHODS: Retrospective study 50 eyes (50 patients) with RRD with unseen retinal breaks in the pre and intraoperative examination. These patients were treated between 2005 and 2010 by vitrectomy or scleral buckling. Retinal breaks were meticulously sought by indentation of the vitreous base. The subretinal fluid was drained by a peripheral retinotomy when a vitrectomy was needed and puncture ab externo when a scleral buckling was performed. RESULTS: A retinal detachment with unseen retinal breaks accounted for 15% of all RRD operated during this 5-year duration period (2005 - 2010). The average age of our patients was 57 years.Ten were myopic (20%) and 27 (54%) pseudophakic, with inferior RRD in 60% of the cases cases while advanced vitreoretinal proliferation (PVR) greater or equal to stage C in was present in 72%. Primary vitrectomy was performed in 46 cases. Retinal reattachment rate was achieved after a single procedure in 41 eyes (82%). Among them, 40 were operated by vitrectomy and one eye by scleral buckling. The recurrence rate was significantly higher in patients operated by scleral buckling (75%) than by vitrectomy (15%). CONCLUSION: RRD with unseen retinal breaks are often seen inferiorly and have a chronic evolution (60%). They concern pseudophakic patients in the majority of the cases. Their poor prognosis and high recurrence rate also appear to be related to an advanced PVR (72%). The good results of primary vitrectomy should be confirmed by randomized studies, especially in phakic eyes.

[Clinical characteristics and therapeutic challenges of retinal detachment in highly myopic eyes]. / Particularités cliniques et thérapeutiques du décollement de rétine du myope fort: à propos de 83 cas.

PURPOSE: To evaluate the clinical characteristics and therapeutic challenges of retinal detachment in highly myopic eyes. PATIENTS AND METHODS: We retrospectively reviewed the medical records of 83 eyes in 79 patients with high myopia (> 6.00 diopters or axial length > or = 26.00 mm) who underwent surgery for retinal detachment between 2001 and 2008. The surgical approaches depended on the type and location of the retinal break, the degree of myopia, and the grade of PVR. RESULTS: The mean age of patients (48 men and 31 women) was 53.9 years. Refractive error ranged from - 10 D to - 25 D (mean was - 14.0 D). The mean follow-up was 19.4 months. Peripheral or equatorial retinal tears were present in 52 cases (62.6%), a macular hole in 14 cases (16.8%), a giant retinal tear in 6 cases (7.2%), and posterior paravascular retinal tears in 11 cases (13.2%). Single-surgery anatomic success was achieved in 65 cases (78.3%), with 17 cases after scleral buckle surgery and 46 cases after pars plana vitrectomy. Final anatomic success was achieved in 76 cases (91.5%). Per and postoperative hemorrhagic complications occurred in 16 cases (19.2%). CONCLUSION: Retinal detachment is a serious complication of high myopia. It often occurs in young patients. Treatment is difficult due to anatomical and clinical conditions.

Characterization of the expression of HTm4 (MS4A3), a cell cycle regulator, in human peripheral blood cells and normal and malignant tissues.

HTm4 (MS4A3) is a member of a family of four-transmembrane proteins designated MS4A. MS4A proteins fulfil diverse functions, acting as cell surface signalling molecules and intracellular adapter proteins. Early reports demonstrated that HTm4 is largely restricted to the haematopoietic lineage, and is involved in cell cycle control, via a regulatory interaction with the kinase-associated phosphatase, cyclin A and cyclin-dependent kinase 2 (CDK2). Here we describe the expression pattern of HTm4 in peripheral blood cells using gene expression microarray technology, and in normal foetal and adult human tissues, as well as adult human cancers, using tissue microarray technology. Using oligonucleotide microarrays to evaluate HTm4 mRNA, all peripheral blood cell types demonstrated very low levels of HTm4 expression; however, HTm4 expression was greatest in basophils compared to eosinophils, which showed lower levels of HTm4 expression. Very weak HTm4 expression is found in monocytes, granulocytes and B cells, but not in T cells, by lineage specific haematopoietic cell flow cytometry analysis. Interestingly, phytohaemagglutinin stimulation increases HTm4 protein expression in peripheral blood CD4-T-lymphocytes over nearly undetectable baseline levels. Western blotting and immunohistochemical studies show strong HTm4 expression in the developing haematopoietic cells of human foetal liver. Immunohistochemical studies on normal tissue microarrays confirmed HTm4 expression in a subset of leucocytes in nodal, splenic tissues and thymic tissue, and weak staining in small numbers of cell types in non-haematopoietic tissues. Human foetal brain specimens from 19 to 31 gestational weeks showed that the strongest-staining cells are ventricular zone cells and the earliest-born, earliest-differentiating 'pioneer' neurons in the cortical plate, Cajal-Retzius and, to a lesser extent, subplate-like neurons. Malignant tissue microarray analysis showed HTm4 expression in a wide variety of adenocarcinomas, including breast, prostate and ovarian. These findings warrant the further study of the role of HTm4 in the cell cycle of both haematopoietic and tumour cells.

Genetic diagnosis in consanguineous families with kidney disease by homozygosity mapping coupled with whole-exome sequencing.

BACKGROUND: Accurate diagnosis of the primary cause of an individual's kidney disease can be essential for proper management. Some kidney diseases have overlapping histopathologic features despite being caused by defects in different genes. In this report, we describe 2 consanguineous Saudi Arabian families in which individuals presented with kidney failure and mixed clinical and histologic features initially believed to be consistent with focal segmental glomerulosclerosis. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: We studied members of 2 apparently unrelated families from Saudi Arabia with kidney disease. MEASUREMENTS: Whole-genome single-nucleotide polymorphism analysis followed by targeted isolation and sequencing of exons using genomic DNA samples from affected members of these families, followed by additional focused genotyping and sequence analysis. RESULTS: The 2 apparently unrelated families shared a region of homozygosity on chromosome 2q13. Exome sequence from affected individuals lacked sequence reads from the NPHP1 gene, which is located within this homozygous region. Additional polymerase chain reaction-based genotyping confirmed that affected individuals had NPHP1 deletions, rather than defects in a known focal segmental glomerulosclerosis-associated gene. LIMITATIONS: The methods used here may not result in a clear genetic diagnosis in many cases of apparent familial kidney disease. CONCLUSIONS: This analysis shows the power of new high-throughput genotyping and sequencing technologies to aid in the rapid genetic diagnosis of individuals with an inherited form of kidney disease. We believe it is likely that such tools may become useful clinical genetic tools and alter the manner in which diagnoses are made in nephrology.

Immunomodulatory function of bone marrow-derived mesenchymal stem cells in experimental autoimmune type 1 diabetes.

Human clinical trials in type 1 diabetes (T1D) patients using mesenchymal stem cells (MSC) are presently underway without prior validation in a mouse model for the disease. In response to this void, we characterized bone marrow-derived murine MSC for their ability to modulate immune responses in the context of T1D, as represented in NOD mice. In comparison to NOD mice, BALB/c-MSC mice were found to express higher levels of the negative costimulatory molecule PD-L1 and to promote a shift toward Th2-like responses in treated NOD mice. In addition, transfer of MSC from resistant strains (i.e., nonobese resistant mice or BALB/c), but not from NOD mice, delayed the onset of diabetes when administered to prediabetic NOD mice. The number of BALB/c-MSC trafficking to the pancreatic lymph nodes of NOD mice was higher than in NOD mice provided autologous NOD-MSC. Administration of BALB/c-MSC temporarily resulted in reversal of hyperglycemia in 90% of NOD mice (p = 0.002). Transfer of autologous NOD-MSC imparted no such therapeutic benefit. We also noted soft tissue and visceral tumors in NOD-MSC-treated mice, which were uniquely observed in this setting (i.e., no tumors were present with BALB/c- or nonobese resistant mice-MSC transfer). The importance of this observation remains to be explored in humans, as inbred mice such as NOD may be more susceptible to tumor formation. These data provide important preclinical data supporting the basis for further development of allogeneic MSC-based therapies for T1D and, potentially, for other autoimmune disorders.

A TRPV2-PKA signaling module for transduction of physical stimuli in mast cells.

Cutaneous mast cell responses to physical (thermal, mechanical, or osmotic) stimuli underlie the pathology of physical urticarias. In vitro experiments suggest that mast cells respond directly to these stimuli, implying that a signaling mechanism couples functional responses to physical inputs in mast cells. We asked whether transient receptor potential (vanilloid) (TRPV) cation channels were present and functionally coupled to signaling pathways in mast cells, since expression of this channel subfamily confers sensitivity to thermal, osmotic, and pressure inputs. Transcripts for a range of TRPVs were detected in mast cells, and we report the expression, surface localization, and oligomerization of TRPV2 protein subunits in these cells. We describe the functional coupling of TRPV2 protein to calcium fluxes and proinflammatory degranulation events in mast cells. In addition, we describe a novel protein kinase A (PKA)-dependent signaling module, containing PKA and a putative A kinase adapter protein, Acyl CoA binding domain protein (ACBD)3, that interacts with TRPV2 in mast cells. We propose that regulated phosphorylation by PKA may be a common pathway for TRPV modulation.

Assessment by miRNA microarray of an autologous cancer antigen-pulsed adoptive immune ensemble cell therapy (AC-ACT) approach; demonstrated induction of anti-oncogenic and anti-PD-L1 miRNAs.

A 60-year-old woman with stage IV rectal cancer received adoptive cell therapy with autologous cancer antigen (AC-ACT) causing induction of anti-oncogenic and anti-PD-L1 miRNAs as assessed by miRNA microarray. More than 1 year after AC-ACT, metastases have been arrested, and the patient reports good quality of life.

Human HTm4 is a hematopoietic cell cycle regulator.

Proper control of cell cycle progression is critical for the constant self-renewal, differentiation, and homeostasis of the hematopoietic system. Cells of all types share the common cell cycle regulators. The different expression patterns of common regulators, in a broad sense, define cell-type or lineage specificity. However, there remains the possibility of hematopoietic cell cycle regulators tailored to the demands of the hematopoietic system. Here we describe a novel protein, HTm4, which serves as a hematopoietic cell cycle regulator. Our data indicate that HTm4 is expressed in hematopoietic tissues and is tightly regulated during the differentiation of hematopoietic stem cells. It binds to cyclin-dependent kinase-associated (CDK-associated) phosphatase-CDK2 (KAP-CDK2) complexes, and the three proteins demonstrate similar patterns of cellular expression in human lymphoid tissues. HTm4 stimulates the phosphatase activity of KAP, and its C-terminal region is required for binding to KAP-CDK2 complexes and the modulation of KAP activity. Overexpression of HTm4 can cause cell cycle arrest at the G(0)/G(1) phase. Thus, HTm4 is a novel hematopoietic modulator for the G(1)-S cell cycle transition.

TRPA1 is a substrate for de-ubiquitination by the tumor suppressor CYLD.

Certain TRP cation channels confer the ability to sense environmental stimuli (heat, cold, pressure, osmolarity) across physiological and pathophysiological ranges. TRPA1 is a TRP-related channel that responds to cold temperatures, and pungent compounds that include the cold-mimetic icilin and cannabinoids. The initial report of TRPA1 as a transformation-associated gene product in lung epithelia is at odds with subsequent descriptions of a tissue distribution for TRPA1 that is restricted to sensory neurons. Here, we report that the human TRPA1 protein is widely expressed outside the CNS, and is indeed dys-regulated during oncogenic transformation. We describe that TRPA1 associates with the tumor-suppressor protein CYLD. TRPA1 is a novel substrate for the de-ubiquitinating activity of CYLD, and this de-ubiquitination has the net effect of increasing the cellular pool of TRPA1 proteins. Oncogenic mutations in the CYLD gene may therefore be predicted to alter cellular levels of TRPA1.

Anti-inflammatory potential of CB1-mediated cAMP elevation in mast cells.

Cannabinoids are broadly immunosuppressive, and anti-inflammatory properties have been reported for certain marijuana constituents and endogenously produced cannabinoids. The CB2 cannabinoid receptor is an established constituent of immune system cells, and we have recently established that the CB1 cannabinoid receptor is expressed in mast cells. In the present study, we sought to define a role for CB1 in mast cells and to identify the signalling pathways that may mediate the suppressive effects of CB1 ligation on mast cell activation. Our results show that CB1 and CB2 mediate diametrically opposed effects on cAMP levels in mast cells. The observed long-term stimulation of cAMP levels by the Galpha(i/o)-coupled CB1 is paradoxical, and our results indicate that it may be attributed to CB1-mediated transcriptional regulation of specific adenylate cyclase isoenzymes that exhibit superactivatable kinetics. Taken together, these results reveal the complexity in signalling of natively co-expressed cannabinoid receptors and suggest that some anti-inflammatory effects of CB1 ligands may be attributable to sustained cAMP elevation that, in turn, causes suppression of mast cell degranulation.

[Characteristics of retinal detachment following neodymium YAG laser posterior capsulotomy: clinical and therapeutic aspects]. / Les décollements de rétine rhegmatogènes post-capsulotomie postérieure au laser YAG: aspects cliniques et thérapeutiques.

PURPOSE: To study the clinical characteristics and the therapeutic results of retinal detachment following neodymium YAG laser posterior capsulotomy. MATERIAL AND METHODS: A retrospective study of 28 patients (28 eyes) operated in department B of the Hédi Rais Institute of Ophthalmology (Tunis, Tunisia) between march 1993 and january 2005 for retinal detachment following neodymium YAG laser posterior capsulotomy. We study the preoperative clinical characteristics of the retinal detachment and the anatomic and functionnal results. RESULTS: The mean age of the patients is 53.4 years. Fourteen patients are men and eleven have high myopia. Ten patients are aphakic and eighteen pseudophakic. The retinal detachment is total or subtotal in 19 eyes (67.9%). The macula is detached in 27 cases (96.4%). The retinal tears are posterior in 9 cases (paravascular retinal break or macular hole) and peripheral in fifteen cases. In 4 cases no tear is found. Vitreoretinal proliferation is advanced in 10 cases. Eleven patients underwent episcleral surgery and 17 endocular surgery. The final success rate is 89%. The postoperative visual acuity increases in 20 cases. It is superior to 1/10 in 14 cases. The minimum follow-up is 6 months. CONCLUSION: The retinal detachment after neodymium laser posterior capsulotomy is serious due to severe vitreoretinal proliferation wich influences the anatomic results.

[Cysts of the iris in an infant. 2 case reports]. / Kystes de l'iris chez l'enfant. A propos de 2 cas.

Iris cysts are rare in children. They might be primary or secondary. Their treatment depends on their type and clinical evolution. The purpose of our study is to discuss the etiologies and therapeutic modalities of iris cysts.

[Foveal choroidal thickness assessment with SD-OCT in high myopic glaucoma]. / Épaisseur choroïdienne fovéolaire en OCT-SD dans le glaucome du myope fort.

PURPOSE: To measure macular choroidal thickness (CT) using spectral domain optical coherence tomography (OCT) in high myopic eyes with primary angle-open glaucoma (POAG), and to investigate whether the choroid is thinner in these eyes compared to high myopic eyes without glaucoma. PATIENTS AND METHODS: We conducted a cross-sectional study of forty-eight eyes with high myopic glaucoma matched with 48 highly myopic eyes without glaucoma by age, central corneal thickness and axial length (AL). OCT scans were performed with the spectral domain OCT (Topcon 2000). The subfoveal CT was measured between the Bruch membrane and the internal aspect of the sclera. RESULTS: In the subgroup without glaucoma, matched with the subgroup with glaucoma (P=0.57), by age, central corneal thickness (P=0.33) and AL (P=0.10), the mean subfoveal CT was 96.32 µm ± 39.56 µm. In the subgroup with glaucoma, the mean subfoveal CT was 50.44 µm ± 16.36 µm. The comparison between the two subgroups found a statistically significant difference in subfoveal CT (P<10(-4)). CONCLUSIONS: Foveal choroidal thickness is reduced in highly myopic eyes with glaucoma. The choroidal thinning can be a useful parameter for the diagnosis and the follow-up of highly myopic patients with glaucoma.

[Microbiological profile of endophthalmitis in a referral centre in Tunisia]. / Profil microbiologique des endophtalmies aiguës dans un centre de référence en Tunisie.

PURPOSE: To investigate the microbiological profile of acute postoperative endophthalmitis in a referral center in Tunisia and to assess the antibiotic sensitivity of the organisms. PATIENTS AND METHODS: This is a retrospective study over a period of eleven years, conducted on patients hospitalized with acute infectious postoperative endophthalmitis. Cultures were performed on aqueous (93%) and vitreous specimens (68%) obtained at presentation. Each sample underwent direct examination, culture and antibiotic susceptibilities. RESULTS: The number of acute postoperative endophthalmitis cases identified during the study period was 308. Organisms were found in 43% of samples (endophthalmitis was bacterial in 39.5%, fungal in 0.9% and polymicrobial in 2.6%). Cultures grew primarily Staphylococcus epidermidis in 31.4% of cases, Streptococcus pneumoniae in 22.7% of cases and Staphylococcus aureus in 12.7% of cases. Gram-positive cocci are more sensitive to vancomycin and Gram-negative bacilli are more susceptible to ofloxacin and ciprofloxacin. CONCLUSION: In our study, microbiological samples were positive in 43% and coagulase-negative Gram-positive cocci are the most common organisms. However, antibiotic resistance has been increasing over the years.

[Simultaneous bilateral rhegmatogenous retinal detachment. 7 case studies]. / Les décollements de rétine rhegmatogènes bilatéraux simultanés. A propos de 7 observations.

PURPOSE: Simultaneous bilateral rhegmatogenous retinal detachments are rare. The purpose of our study was to examine the incidence, predictive factors, surgical results and prognosis of these retinal detachments. PATIENTS AND METHODS: A retrospective analysis of the medical records of 468 consecutive patients, 7-89 years of age (mean, 45.7 years), undergoing surgery for rhegmatogenous retinal detachment between 1993 and 2000. RESULTS: During this period, a total of 497 operations for rhegmatogenous retinal detachment were done. Thirty-two patients had bilateral rhegmatogenous retinal detachment. Simultaneous detachments were observed in seven patients (1.5%). In four cases, the diagnosis of retinal detachment was fortuitous. In fact, most patients presented with unilateral symptoms. The mean age (35 years) of patients suffering from simultaneous bilateral rhegmatogenous retinal detachment was younger than that of patients with unilateral or consecutive bilateral retinal detachments. Five patients were myopic. Multiple round retinal holes were the most frequent lesions responsible for retinal detachment. A preoperative proliferative vitreoretinopathy was found in 57% of cases. The retina was reattached in nine cases (81%). CONCLUSION: Simultaneous bilateral rhegmatogenous retinal detachment is usually found in relatively young myopic patients with atrophic retinal holes. They are rare but severe because of their frequent association with preoperative proliferative vitreoretinopathy.