Laminar fMRI and computational theories of brain function.

Recently developed methods for functional MRI at the resolution of cortical layers (laminar fMRI) offer a novel window into neurophysiological mechanisms of cortical activity. Beyond physiology, laminar fMRI also offers an unprecedented opportunity to test influential theories of brain function. Specifically, hierarchical Bayesian theories of brain function, such as predictive coding, assign specific computational roles to different cortical layers. Combined with computational models, laminar fMRI offers a unique opportunity to test these proposals noninvasively in humans. This review provides a brief overview of predictive coding and related hierarchical Bayesian theories, summarises their predictions with regard to layered cortical computations, examines how these predictions could be tested by laminar fMRI, and considers methodological challenges. We conclude by discussing the potential of laminar fMRI for clinically useful computational assays of layer-specific information processing.

THE NASOLABIAL FLAP: THE MOST VERSATILE METHOD IN FACIAL RECONSTRUCTION.

The nasolabial flap was described 170 years ago and still remains one of the most frequently used methods in facial reconstruction. This technically easy and maximally effective procedure has become a real workhorse and an integral instrument for every plastic surgeon. Over time multiple modifications of this technique have been described. In this article, authors present an overview of nasolabial flap modalities and discuss advantages and disadvantages of these techniques.

SURGICAL TREATMENT OF MELANOMA.

Malignant melanoma is a serious disease, the incidence of which rises. Since the most important treatment method is sufficient wide skin and subcutaneous tissue excision, plastic surgeon is often the only specialist who is able to close the resulting defect. This paper deals with recommendations and treatment options for malignant melanoma from the point of a plastic surgeon. The primary width of excised safety rim of healthy tissue with regards to the depth of melanoma invasion differs. Safety margin is 0.5 cm for melanoma in situ, 1 cm in Breslow up to 2 mm and 2 cm in Breslow over 2 mm. Furthermore, there is indication for sentinel lymph node biopsy, which should be performed in melanoma with Breslow over 1 mm, and in risky melanoma in Breslow above 0.75 mm. Every patient with stage IIB malignant melanoma and above according to TNM classification should undergo adjuvant therapy in a specialized centre. Ideal condition for the patient is a permanent and close cooperation between a dermatologist, oncologist and plastic surgeon, who supplement each other in diagnostics, therapy and follow up of the patients.

[Influencing factors and complications in open treatment of acute anterior glenoid rim fractures]. / Einflussfaktoren und Komplikationen bei offener Versorgung akuter anteriorer Glenoidrandfrakturen.

BACKGROUND: The aim of this comparative study was to evaluate the clinical radiological outcome after open treatment of acute anterior glenoid rim fractures and to analyze the influencing factors and complications. PATIENTS AND METHODS: The study included 26 patients with an average age of 51.6 years (range 27-71 years) at surgery. The mean period of follow-up was 5.1 years (range 2.0-11.1 years). The average extent of glenoid fracture involvement was 25.5 % (range 18-35%) and three fixation techniques were applied: 1) bioresorbable pins (n = 16), 2) small fragment screws (n = 5) and 3) bioresorbable suture anchors (n = 5). RESULTS: The mean score values were 80.3 points for the absolute Constant score, 87.6 points for the normalized Constant score, 88.7 points for the Rowe score, 17.4 points for the Oxford shoulder score, 10.3 points for the simple shoulder test, 13.0 points for the shoulder pain and disability index and 81.5 % for the subjective shoulder value. The fixation technique did not show a significant influence; however, multi-fragment fractures were associated with a significantly inferior absolute (73 vs. 87 points, p = 0.022) and normalized Constant scores (81 vs. 94 points, p = 0.019). Subscapularis insufficiency with internal rotation deficit was found in 10 (39 %) patients and posttraumatic osteoarthritis occurred in 6 (23 %) patients. CONCLUSION: Open fixation yielded good or excellent shoulder function in 20 out of the 26 (77 %) patients and the clinical outcome primarily depended on the underlying type of fracture. Significantly inferior outcomes should be expected in patients with multi-fragment fractures. The main complications were subscapularis insufficiency and posttraumatic osteoarthritis.

Structure-function-behavior relationship in estrogen-induced synaptic plasticity.

This article is part of a Special Issue "Estradiol and Cognition". In estrogen-induced synaptic plasticity, a correlation of structure, function and behavior in the hippocampus has been widely established. 17ß-estradiol has been shown to increase dendritic spine density on hippocampal neurons and is accompanied by enhanced long-term potentiation and improved performance of animals in hippocampus-dependent memory tests. After inhibition of aromatase, the final enzyme of estradiol synthesis, with letrozole we consistently found a strong and significant impairment of long-term potentiation (LTP) in female mice as early as after six hours of treatment. LTP impairment was followed by loss of hippocampal spine synapses in the hippocampal CA1 area. Interestingly, these effects were not found in male animals. In the Morris water maze test, chronic administration of letrozole did not alter spatial learning and memory in either female or male mice. In humans, analogous effects of estradiol on hippocampal morphology and physiology were observed using neuroimaging techniques. However, similar to our findings in mice, an effect of estradiol on memory performance has not been consistently observed.

Is Whole-Body Computed Tomography the Standard Work-up for Severely-Injured Children? Results of a Survey among German Trauma Centers.

PURPOSE OF THE STUDY: Whole-body computed tomography is accepted as the standard procedure in the primary diagnostic of polytraumatised adults in the emergency room. Up to now there is still controversial discussion about the same algorithm in the primary diagnostic of children. The aim of this study was to survey the participation of German trauma-centres in the care of polytraumatised children and the hospital dependant use of whole-body computed tomography for initial patient work-up. MATERIAL AND METHODS: A questionnaire was mailed to every Department of Traumatology registered in the DGU (German Trauma Society) databank. RESULTS: We received 60,32% of the initially sent questionnaires and after applying exclusion criteria 269 (53,91%) were applicable to statistical analysis. In the three-tiered German hospital system no statistical difference was seen in the general participation of children polytrauma care between hospitals of different tiers (p = 0.315). Even at the lowest hospital level 69,47% of hospitals stated to participate in polytrauma care for children, at the intermediate and highest level hospitals 91,89% and 95,24% stated to be involved in children polytrauma care, respectively. Children suspicious of multiple injuries or polytrauma received significantly fewer primary whole-body CTs in lowest level compared to intermediate level hospitals (36,07% vs. 56,57%; p = 0.015) and lowest level compared to highest level hospitals (36,07% vs. 68,42%; p = 0.001). Comparing the use of whole-body CT in intermediate to highest level hospitals a not significant increase in its use could be seen in highest level hospitals (56,57% vs. 68,42%; p = 0.174). CONCLUSION: According to our survey, taking care of polytraumatised children in Germany is not limited to specialised hospitals or a defined hospital level-of-care. Additionally, there is no established radiologic standard in work-up of the polytraumatised child. However, in higher hospital care -levels a higher percentage of hospitals employs whole-body CTs for primary radiologic diagnostics in polytraumatised children.

Catastrophe risk models for evaluating disaster risk reduction investments in developing countries.

Major natural disasters in recent years have had high human and economic costs, and triggered record high postdisaster relief from governments and international donors. Given the current economic situation worldwide, selecting the most effective disaster risk reduction (DRR) measures is critical. This is especially the case for low- and middle-income countries, which have suffered disproportionally more economic and human losses from disasters. This article discusses a methodology that makes use of advanced probabilistic catastrophe models to estimate benefits of DRR measures. We apply such newly developed models to generate estimates for hurricane risk on residential structures on the island of St. Lucia, and earthquake risk on residential structures in Istanbul, Turkey, as two illustrative case studies. The costs and economic benefits for selected risk reduction measures are estimated taking account of hazard, exposure, and vulnerability. We conclude by emphasizing the advantages and challenges of catastrophe model-based cost-benefit analyses for DRR in developing countries.

Penis augmentation by application of silicone material: complications and surgical treatment.

BACKGROUND: Complications resulting from enlargement of the penis by applications of unknown types of silicone and mineral oils are well described. Surgical removal of the tissue altered by inflammation leads to the development of defects of various sizes, often circular from the glans penis to the scrotum. The options of subsequent surgical treatment described in literature are not very extensive. Most defects are managed with skin grafting, rarely V-Y advancement or bilateral scrotal flaps. METHODS: We present a 36-year-old patient after application of unknown silicone material into the penis for cosmetic enlargement. After the application developed severe inflammation with ulceration and necrosis around the penis. Conservative treatment was not effective, therefore, the infiltrated skin with subcutaneous tissue of the entire penis was surgically removed. The resulting defect was covered by implantation of the penis under the skin of the scrotum. RESULT: There were no complications in the postoperative course, pain that was present before the surgery immediately subsided. Skin suture healed completely within 14 days. Three months after the surgery the patient returned to normal sexual life. CONCLUSIONS: Implantation of the penis under the skin of the scrotum is a fast, safe and effective method that can treat most of the circular skin defects of the penis. Scrotal skin is thin, soft, elastic and creates abundant and good cover around the entire penis.

The interpolation nasolabial flap: the advantageous solution for nasal tip reconstruction in elderly and polymorbid patients.

BACKGROUND: Nasal tip is one of the most frequent areas affected by basocellular carcinoma. The treatment must avoid any postsurgical deformity, as the nasal tip is the main part of the facial triangle. Most of these patients are elderly and polymorbid. The surgeon must find a compromise between the cosmetic effect and an acceptable stress for the patient. METHODS: There were five methods of nasal tip reconstruction used on a group of fifty elderly patients with various medical comorbidities: skin graft, bilobed flap, dorsal nasal flap, forehead flap and interpolation nasolabial flap. The resulting cosmetic effect was objectivized by a standardized questionnaire and the advantages and disadvantages of each method were discussed. RESULTS: According to the characteristics that were considered (good vascular supply, matching skin color and texture, sufficient amount of material, possibility to cover exposed cartilage or to combine it with cartilaginous graft; scars of minimal visibility, perfect aesthetic outcome, use of local anesthesia, short surgical time, and technical simplicity) was the interpolation nasolabial flap found as the most appropriate. CONCLUSION: Interpolation nasolabial flap is an easy and effective method for treatment of smaller and middle-sized defects of the nasal tip with minimal stress for the patient. LEVEL OF EVIDENCE: Level III.

The SPEAK study rationale and design: A linguistic corpus-based approach to understanding thought disorder.

AIM: Psychotic symptoms are typically measured using clinical ratings, but more objective and sensitive metrics are needed. Hence, we will assess thought disorder using the Research Domain Criteria (RDoC) heuristic for language production, and its recommended paradigm of "linguistic corpus-based analyses of language output". Positive thought disorder (e.g., tangentiality and derailment) can be assessed using word-embedding approaches that assess semantic coherence, whereas negative thought disorder (e.g., concreteness, poverty of speech) can be assessed using part-of-speech (POS) tagging to assess syntactic complexity. We aim to establish convergent validity of automated linguistic metrics with clinical ratings, assess normative demographic variance, determine cognitive and functional correlates, and replicate their predictive power for psychosis transition among at-risk youths. METHODS: This study will assess language production in 450 English-speaking individuals in Australia and Canada, who have recent onset psychosis, are at clinical high risk (CHR) for psychosis, or who are healthy volunteers, all well-characterized for cognition, function and symptoms. Speech will be elicited using open-ended interviews. Audio files will be transcribed and preprocessed for automated natural language processing (NLP) analyses of coherence and complexity. Data analyses include canonical correlation, multivariate linear regression with regularization, and machine-learning classification of group status and psychosis outcome. CONCLUSIONS: This prospective study aims to characterize language disturbance across stages of psychosis using computational approaches, including psychometric properties, normative variance and clinical correlates, important for biomarker development. SPEAK will create a large archive of language data available to other investigators, a rich resource for the field.

A Review of the EUSO-Balloon Pathfinder for the JEM-EUSO Program.

EUSO-Balloon is a pathfinder for JEM-EUSO, the mission concept of a spaceborne observatory which is designed to observe Ultra-High Energy Cosmic Ray (UHECR)-induced Extensive Air Showers (EAS) by detecting their UltraViolet (UV) light tracks "from above." On August 25, 2014, EUSO-Balloon was launched from Timmins Stratospheric Balloon Base (Ontario, Canada) by the balloon division of the French Space Agency CNES. After reaching a floating altitude of 38 km, EUSO-Balloon imaged the UV light in the wavelength range ∼290-500 nm for more than 5 hours using the key technologies of JEM-EUSO. The flight allowed a good understanding of the performance of the detector to be developed, giving insights into possible improvements to be applied to future missions. A detailed measurement of the photoelectron counts in different atmospheric and ground conditions was achieved. By means of the simulation of the instrument response and by assuming atmospheric models, the absolute intensity of diffuse light was estimated. The instrument detected hundreds of laser tracks with similar characteristics to EASs shot by a helicopter flying underneath. These are the first recorded laser tracks measured from a fluorescence detector looking down on the atmosphere. The reconstruction of the direction of the laser tracks was performed. In this work, a review of the main results obtained by EUSO-Balloon is presented as well as implications for future space-based observations of UHECRs.

Nasal reconstruction in children with the combination of nasolabial and island flaps.

BACKGROUND: The reconstruction of the nose is one of the most complicated aesthetic-reconstructive procedures. The difficulty of the procedure lies is in the necessity for reconstruction not only to capture the very complicated, various shapes of the nose but also to preserve the function of the nose: to allow the patient to breathe through the nose. MATERIALS AND METHODS: 12-year-old girl had loss injury of the part of left wing of the nostril. We used the compound nasolabial flap with a small excess to resolve the mucosal and skin defect. One year after the first operation relief of natural transition of the new wing of the nostril and cheek was created with small island flap. RESULTS AND CONCLUSIONS: The reconstruction of a wing of the nostril in multistage procedures with combined nasolabial flap and island flap allowed us to perform precise modelation of the nostril wing with the natural transition to the cheek. An island flap with its scars creates the required contour of a nostril wing and prevents the collapse and flattening of the nostril wing externally.

Thawing the frozen shoulder. A randomised trial comparing manipulation under anaesthesia with hydrodilatation.

This study prospectively evaluated the outcome of manipulation under anaesthesia and hydrodilatation as treatments for adhesive capsulitis. A total of 36 patients (38 shoulders) were randomised to receive either method, with all patients being treated in stage II of the disease process. The mean age of the patients was 55.2 years (44 to 70) and the mean duration of symptoms was 33.7 weeks (12 to 76). Eighteen shoulders (17 patients) underwent manipulation under anaesthesia and 20 (19 patients) had hydrodilatation. There were three insulin-dependent diabetics in each group. The mean visual analogue score in the manipulation under anaesthesia group was 5.7 (3 to 8.5; n = 18) before treatment, 4.7 (0 to 8.5; n = 16) at two months (paired t-test p = 0.02), and 2.7 (0 to 9; n = 16) at six months (paired t-test, p = 0.0006). The mean score in the hydrodilatation group was 6.1 (4 to 10; n = 20) before treatment, 2.4 (0 to 8; n = 18) at two months (paired t-test, p = 0.001), and 1.7 (0 to 7; n = 18) at six months (paired t-test, p = 0.0006). The visual analogue scores in the hydrodilatation group were significantly better than in the manipulation under anaesthesia group over the six-month follow-up period (p < 0.0001). The mean Constant score in those manipulated was 36 (26 to 66) before treatment, 58.5 (24 to 90) at two months (paired t-test, p = 0.001) and 59.5 (23 to 85) at six months (paired t-test, p = 0.0006). In the hydrodilatation group it was 28.8 (18 to 55) before treatment, 57.4 (17 to 80) at two months (paired t-test, p = 0.0004) and 65.9 (28 to 92) at six months (paired t-test, p = 0.0005). The Constant scores in the hydrodilatation group were significantly better than in the manipulated group over the six-month period of follow-up (p = 0.02). The range of movement improved in all patients over the six months, but was not significantly different between the groups. At the final follow-up, 94% of patients (17 of 18) were satisfied or very satisfied after hydrodilatation compared with 81% (13 of 16) of those receiving a manipulation. Most of our patients were treated successfully, but those undergoing hydrodilatation did better than those who were manipulated.

Severity-dependent differences in early management of thoracic trauma in severely injured patients - Analysis based on the TraumaRegister DGU®.

BACKGROUND: Major trauma is associated with chest injuries in nearly 50% of multiple injuries. Thoracic trauma is a relevant source of comorbidity throughout the period of multiply-injured patient care and may require swift and well-thought-out interventions in order to avert a deleterious outcome. In this epidemiological study we seek to characterize groups of different thoracic trauma severity in severely injured patients and identify related differences in prehospital and early clinical management. This may help to anticipate necessary treatment for chest injuries. METHODS: Patients documented between 2002 and 2012 in the TraumaRegister DGU®, aged ≥ 16 years, determined Injury Severity Score ≥ 16, and documentation from European trauma centers were analyzed. Isolated brain injury and severe head injury (Abbreviated Injury ScaleHead ≥ 4) led to patient exclusion. Patient subgroups were formed according to the Abbreviated Injury ScaleThorax as Controls, AIS-2, AIS-3, AIS-4, and AIS-5/6. Demographic and clinical characteristics comparing the aforementioned groups were evaluated using descriptive statistics. RESULTS: Twenty two thousand five hundred sixty five predominantly male (74%) patients, mean age 45.7 years (SD 19.3), suffering from blunt trauma (95%), and presenting a mean Injury Severity Score of 25.6 (SD 9.6) were analyzed. Higher thoracic injury severity was associated with more different thoracic injuries. The highest rate of prehospital intubation (58%) occurred in AISThorax-5/6. The worse the chest trauma, the more chest tubes were placed prehospitally, peaking at 22% in AISThorax-5/6. Out-of-hospital cardiopulmonary resuscitation was successfully performed in 11% in AISThorax-5/6 compared to 1%-3% in lesser thoracic trauma severity. Massive transfusion and emergency surgery was highest in AISThorax-5/6 compared to lesser thoracic injury (12% vs. 5%-7% and 17% vs. 3%-7%) and both were independently associated with thoracic injuries in patients with AISThorax ≥ 4. CONCLUSIONS: We provide epidemiological data on trauma mechanism, concomitant injuries, frequencies of emergency interventions and outcome associated with different thoracic trauma severity. Prehospital and early clinical management is more complex when AISThorax is ≥ 4. Severely injured patients with critical thoracic trauma are most challenging to take care of with highest rates in prehospital intubation, cardiopulmonary resuscitation, chest tube placements, blood transfusions as well as emergency surgery.

Mechanisms linking electrical alternans and clinical ventricular arrhythmia in human heart failure.

BACKGROUND: Mechanisms of ventricular tachycardia (VT) and ventricular fibrillation (VF) in patients with heart failure (HF) are undefined. OBJECTIVE: The purpose of this study was to elucidate VT/VF mechanisms in HF by using a computational-clinical approach. METHODS: In 53 patients with HF and 18 control patients, we established the relationship between low-amplitude action potential voltage alternans (APV-ALT) during ventricular pacing at near-resting heart rates and VT/VF on long-term follow-up. Mechanisms underlying the transition of APV-ALT to VT/VF, which cannot be ascertained in patients, were dissected with multiscale human ventricular models based on human electrophysiological and magnetic resonance imaging data (control and HF). RESULTS: For patients with APV-ALT k-score >1.7, complex action potential duration (APD) oscillations (≥2.3% of mean APD), rather than APD alternans, most accurately predicted VT/VF during long-term follow-up (+82%; -90% predictive values). In the failing human ventricular models, abnormal sarcoplasmic reticulum (SR) calcium handling caused APV-ALT (>1 mV) during pacing with a cycle length of 550 ms, which transitioned into large magnitude (>100 ms) discordant repolarization time alternans (RT-ALT) at faster rates. This initiated VT/VF (cycle length <400 ms) by steepening apicobasal repolarization (189 ms/mm) until unidirectional conduction block and reentry. Complex APD oscillations resulted from nonstationary discordant RT-ALT. Restoring SR calcium to control levels was antiarrhythmic by terminating electrical alternans. CONCLUSION: APV-ALT and complex APD oscillations at near-resting heart rates in patients with HF are linked to arrhythmogenic discordant RT-ALT. This may enable novel physiologically tailored, bioengineered indices to improve VT/VF risk stratification, where SR calcium handling and spatial apicobasal repolarization are potential therapeutic targets.

Beta-catenin mutations in hepatocellular carcinoma correlate with a low rate of loss of heterozygosity.

To determine the frequency of Wnt/Wingless beta catenin pathway alteration in human hepatocellular carcinoma, a beta catenin and APC gene mutation screening was performed in a series of 119 tumors. An activating beta catenin mutation in exon 3 was found in 18% of the cases. Among tumors lacking beta catenin mutation, no APC mutation has been evidenced in a subset of 30 cases tested. The correlation between beta catenin mutation status and chromosome segment deletions was studied on a set of 48 hyperploid tumors. Chromosome 1p, 4q and 16p deletions were significantly associated with the absence of beta catenin mutation (P<0.05). Furthermore the Fractional Allelic Loss was significantly smaller in the beta catenin mutated tumors than in the non-mutated tumors (0.12 versus 022). Taken together, these results suggest, the existence of two carcinogenesis mechanisms. The first mechanism implies a beta catenin activating mutation associated with a low rate of loss of heterozygosity. The second mechanism, operating in a context of chromosomal instability, would involve tumor suppressor genes.

Identification of homozygous deletions at chromosome 16q23 in aflatoxin B1 exposed hepatocellular carcinoma.

Loss of heterozygosity (LOH) represents the most frequent genetic alteration observed in hepatocellular carcinoma (HCC). Chromosome 16q is of particular interest as it exhibits LOH in 29% of HCC tumors and is frequently lost in breast, prostate, ovarian and gastric carcinomas. We genotyped 157 HCC tumors for 17 microsatellite markers distributed on chromosome 16q and determined a common region of LOH localized between the markers D16S518 and D16S504. By refining the boundaries of two interstitial LOH and two homozygous deletions, the critical region was delimited to 180 kb between D16S3096 and D16S3029. This region is located in intron 8 of the WWOX/FOR gene, but a search for mutations in all coding exons of this gene in 27 HCC tumors and cell lines did not reveal any tumor somatic alterations. Furthermore, by RT-PCR, no abnormal transcripts of this WWOX/FOR gene was detected in nine HCC cell lines. Finally, analysis of the p53 gene mutations with the clinical parameters of all tumors revealed that the two homozygous deletions have occurred in tumors presenting a R249S mutation. Our data revealed a relationship between chromosome 16q homozygous deletions and R249S p53 mutations in tumors where the patient had been exposed to aflatoxin B1 (P=0.002). These results are consistent with a role of aflatoxin B1 in the instability of chromosome 16q at the fragile site FRA16D. However, the nature of the specific gene that is altered during hepatocarcinogenesis remains to be elucidated.

Efficient transduction of hemopoietic CD34+ progenitors of human origin using an original retroviral vector derived from Fr-MuLV-FB29: in vitro assessment.

A novel retroviral vector has been designed based on a Friend-murine leukemia virus (Fr-MuLV) FB29 strain. The latter has been selected according to characteristics of pathogenicity in mice where it induces a disease of the haemopoietic system affecting all lineages. Higher infectivity has also been demonstrated as compared to other strains. In accordance with these findings, the amphotropic producer clone used in this study carrying along the neomycine resistance gene (FOCH-Neo), harbors viral titers over 10(7) cfu/ml. To investigate the potential of genetically engineering hematopoietic precursors, CD34+ progenitors were selected from cord blood, bone marrow, and peripheral blood mobilized stem cells (patients + solid tumors) and transduced with FOCH-Neo. High transduction rates were achieved using virus supernatant and minimal doses of hematopoietic growth factors during pretransduction and transduction steps. A polymerase chain reaction (PCR) assay investigating the presence of both neomycin-encoding and viral vector sequences tested positive in 45-90% of granulocyte-macrophage colony-forming units (CFU-GM) generating cells (bone marrow and peripheral blood derived cells) following transduction. An average of 35% colonies showed resistance to G418. Such levels of transduction proved reproducible using only supernatants harboring over 10(7) cfu/ml. In those experiments where long-term in vitro cultures could be maintained over 5 weeks (all cord blood and 5 among 23 PBSC), efficient transduction of long-term culture initiating cell (LTC-IC) hematopoietic progenitors was demonstrated on the basis of both resistance to G418 and virus integration. In the latter case, the PCR assay tested positive in as much as 35-60% of late unselected CFU-colonies. This novel retroviral vector harbors interesting features toward genetic modification of hematopoietic progenitors.

Culture of hematopoietic stem cells purified from murine bone marrow.

The results of the Y-chromosome in situ hybridization experiments, the MRA assessment, and the long-term production of CFU-GM in vitro indicate that our protocol to sort low density WGA+, 15/1.1-, Rh123 dull cells enriches about 200-fold for PHSC. Assays for spleen colony formation (CFU-S) and radioprotection (30-day survival) were shown to be unspecific for PHSC, and, therefore, we lack a quantitative PHSC assay. The absolute number of PHSC in the bone marrow is not known any more, the purity of our sorted population likewise is unknown. Long-term repopulating cells (PHSC) could be separated from short-term repopulating ones by using Rh123 staining. The short-term repopulating cells (Rh123 bright) provided sufficient offspring to protect lethally irradiated mice until endogenous PHSC could reconstitute hematopoiesis. These cells are therefore of interest for bone marrow transplantation, because they provide radioprotection without long-term repopulation and graft-versus-host disease. For gene therapy these cells are of limited use, and PHSC with extensive replication are needed. The PHSC were not cultured successfully. Less than 15% of the sorted Rh123 dull cells responded in semisolid or liquid cultures in the presence of growth factors. Proliferation without differentiation was not observed. This may indicate that the right growth factor has not been found yet. On the other hand, about 30% of the cells responded in stromal layers of long-term bone marrow cultures and prolonged CFU-GM production and cobblestone area formation were observed there, suggesting that cell-cell contact and adherence molecules play a regulatory role in PHSC replication.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of E. coli exonuclease III to generate single stranded DNA in BrdUrd cell-cycle analysis permits simultaneous detection of cell surface antigens.

An immunocytochemical method for the simultaneous flow cytometric quantitation of total cellular DNA, incorporated 5-bromo-2'-deoxyuridine (BrdUrd) and one or more cell surface antigens has been developed. Biotin labeling of cell surface antigens, critically tuned fixation techniques and an enzymatic denaturation of cellular DNA are the essential features of this method. Enzymatic denaturation of cellular DNA was shown to prevent loss of cell surface antigen-bound biotin moieties, and thus to preserve cell surface immunofluorescence distribution. After a mild protein extraction and the introduction of breaks into the chromatin using restriction endonucleases, E. coli exonuclease III was used to generate stretches of single stranded DNA. This approach permits detection of the incorporated BrdUrd using anti-BrdUrd monoclonal antibodies. The enzymatic denaturation protocol was optimized using in vitro BrdUrd-labeled L1210 murine leukemia cells, and applied to both in vivo and ex vivo BrdUrd-labeled murine bone marrow cells. With this new method it is possible to study DNA content, cell cycle kinetics and cell surface antigen expression simultaneously, and hence functional relationships between these parameters can be investigated.