First use of intraventricular nicardipine in a pediatric patient with vasospasm secondary to meningitis: illustrative case.

BACKGROUND: Cerebral vasospasm is commonly associated with adult aneurysmal subarachnoid hemorrhage but can develop in children. The standard vasospasm treatment includes induced hypertension, avoidance of hypovolemia, systemic use of the calcium channel blocker (CCB) nimodipine, and cerebral angiography for intraarterial therapy. Emerging treatments in adults, such as intraventricular CCB administration, have not been investigated in children. This study demonstrates the successful use of an intraventricular CCB in a pediatric patient with refractory vasospasm secondary to meningitis. OBSERVATIONS: A 12-year-old female presented with Streptococcus pneumoniae meningitis and ventriculitis with refractory symptomatic cerebral vasospasm. She received a 5-day course of intrathecal nicardipine through an existing external ventricular drain. Her clinical status, transcranial Doppler studies, and radiography improved. Treatment was well tolerated. LESSONS: Pediatric vasospasm is uncommon and potentially devastating. The management of vasospasm in adults occurs frequently. Principles of this management are adapted to pediatric care given the rarity of vasospasm in children. The use of intraventricular nicardipine has been reported in the care of adults with level 3 evidence. It has not been adequately reported in children with refractory vasospasm. Here, the first use of intraventricular nicardipine in treating pediatric cerebral vasospasm in the setting of meningitis is described and highlighted.

Postmortem Protocols of Implantable Neurosurgical Devices: A Cross-Sectional Survey.

BACKGROUND: Implantable devices are increasingly more common for management of movement disorders, pain, and epilepsy. These devices are often complex and constructed of nonbiodegradable or hazardous materials. Therefore, proper postmortem handling of these devices is exceedingly important. Unfortunately, there is no consolidated resource available for postmortem neuromodulation device protocols. Thus, we surveyed and catalogued the protocols for implantable devices to summarize proper postmortem device protocols for implantable neurosurgical devices currently on the market. METHODS: We performed a cross-sectional study of companies producing commonly implanted neurosurgical devices. Using information from company websites, user manuals, and catalogs we categorized devices into 3 groups: A (formal recommendation for explantation), B (recommendation for explantation without formal company protocol), and C (explantation is not necessary). We then compiled the data into a stoplight diagram, providing a clear postmortem disposal algorithm for each device category. RESULTS: Twelve companies were queried regarding 46 devices. Postmortem protocols were available for 50% (23/46) of devices; the remaining devices did not have formal recommendations. Overall, 50% of devices were classified as category A "red light" on the stoplight diagram based on recommendations, 10.9% as category B "yellow light," and the remaining 39.1% were classified as category C "green light" indicating they are safe to bury or cremate. CONCLUSIONS: Evolution in therapies and growth in functional neurosurgery has expanded the range of implantable neurosurgical devices. We provide an educational document summarizing their postmortem protocols. This resource aims to aid health-care providers and encourage proper disposal practices during burial or cremation.

Neurosurgery trainee well-being in a pediatric neurosurgery hospital: baseline data to motivate toward implementing change.

OBJECTIVE: The aim of this study was to obtain aggregated baseline pediatric neurosurgery well-being data at a tertiary care institution. METHODS: An institutional grant funded the completion of the Maslach Burnout Inventory (MBI) by 100% (n = 13) of the trainees during a 1-year period, including 1 pediatric neurosurgery fellow and 12 residents from 4 regional neurosurgery training programs. Aggregated and anonymized group results included frequency scores ranging from 0 (never) to 6 (every day). The mean ± SD group scores were compared to the general population of > 11,000 people in the human services professions. Burnout profiles were calculated on the basis of MBI scale scores by using established comparisons to standardized normal values. Burnout profile types include engaged, ineffective, overextended, disengaged, and burnout. RESULTS: The mean ± SD score for emotional exhaustion was 2.6 ± 1.1 for trainees compared with 2.3 ± 1.2 in the comparison population. The mean ± SD score for depersonalization was 1.6 ± 1 compared with 1.7 ± 1.2 in the comparison population. The mean ± SD score for personal accomplishment was 4.9 ± 0.7 compared with 4.3 ± 0.9 in the comparison population. Profiles were classified as engaged (n = 6), ineffective (n = 3), overextended (n = 3), and burnout (n = 1). CONCLUSIONS: Problematic profiles were present for more than half (7 [53.8%]) of pediatric neurosurgery trainees who cited higher emotional exhaustion than the general population of healthcare providers. Trainees scored lower in depersonalization and higher in personal accomplishment compared with the general population, which are both protective against burnout. Targeting factors that contribute to emotional exhaustion may have an impact on improving the overall well-being of pediatric neurosurgery trainees.

Endoscopic placement of a triventricular stent for complex hydrocephalus and isolated fourth ventricle: illustrative case.

BACKGROUND: Hydrocephalus is commonly encountered in pediatric neurosurgery. The etiology is diverse, and complexity in management increases in patients with loculated or trapped ventricles. The authors sought to examine a treatment option of endoscopic placement of a triventricular stent in a pediatric patient with complex hydrocephalus and a trapped fourth ventricle. OBSERVATIONS: In this case, the authors present the treatment of complex hydrocephalus with a trapped fourth ventricle in a pediatric patient using endoscopic placement of a triventricular aqueductal stent. The patient had a complex neurosurgical history, which included over 15 surgeries for shunted hydrocephalus. This case highlights the unique approach used, and the authors discuss surgical nuances of the technique, as well as learning points. LESSONS: Complex hydrocephalus can be difficult to manage because patients often have multiple catheters, loculated or trapped ventricles, and extensive surgical histories. Endoscopic placement of a triventricular stent can decrease shunt system complexity, restore normal cerebrospinal fluid pathway circulation across the cerebral aqueduct, and promote communication between the ventricles. The authors' treatment modality resulted in the successful resolution of the trapped fourth ventricle and symptomatic improvement in hydrocephalus.

Navigated Radiofrequency Ablation Peripheral Rhizotomy for Lumbosacral Hypertonia in a Nonambulatory Patient With Spinal Fusion: Indications, Surgical Techniques, and Lessons Learned.

BACKGROUND AND OBJECTIVES: Radiofrequency ablation (RFA) is a destructive therapy which causes target tissue destruction by application of a thermal dose. Neurosurgical applications of RFA are well-described for myriad chronic pain and movement disorder diagnoses. In fact, RFA pallidotomy and thalamotomy are the initial procedures from which the field of neurosurgical management for movement disorders emerged. RFA rhizotomy for post-traumatic spasms was popular in the 1970s and 1980s, although it was largely abandoned after the invention and Food and Drug Administration approval of intrathecal baclofen therapy. RFA has not been described as a primary treatment of hypertonia in nonambulatory children. METHODS: We report a case of computer-navigated, nonselective RFA peripheral rhizotomy for a nonambulatory child with a history of severe scoliosis and spinal fusion, where an open rhizotomy was technically impractical. RESULTS: Navigation to and ablation of the bilateral L1-L5 peripheral nerves with this approach was successful, and the patient experienced bilateral lower extremity tone improvement. CONCLUSION: We use this case to highlight considerations in indications, our applied operative technique, and lessons learned from this novel application of RFA peripheral rhizotomy in children.

Correlating Thermodynamics with Cognitive Outcomes in Magnetic Resonance-guided Laser Interstitial Thermal Therapy (MRgLITT) of a Pediatric Hypothalamic Hamartoma.

OBJECTIVE: Magnetic resonance-guided laser interstitial thermal therapy (MRgLITT) is a commonly used clinical method of destroying intracranial brain foci. Our objective was to correlate the thermal damage estimate transition zone with cognitive outcomes in MRgLITT of a pediatric hypothalamic hamartoma. METHODS: Uncomplicated MRgLITT was used to disconnect an 8-mm left Delalande grade II hypothalamic hamartoma (HH) revealed on neuroimaging of a 17-year-old male patient with drug-resistant epilepsy and a "gelastic +" semiology including both gelastic and tonic-clonic seizures. Despite meticulous planning, submillimetric stereotactic accuracy, and reassuring intraoperative thermography, the patient experienced transient, but profound, global amnesia. Retroactively, we applied a new iteration of thermographic software that overlays a magenta-colored transition zone (TZ) around the necrotic zone defined by the orange-pigmented thermal damage estimate (TDE). RESULTS: Clear involvement of the bilateral mesial circuits was demonstrated by the overlay of the TZ on the TDE. CONCLUSIONS: Involvement of the bilateral mesial circuits visualized with TDE and TZ could account for the neurocognitive outcomes of our patient. We highlight this case as our understanding of thermography analysis evolves, emphasizing principles of technique and trajectory planning, as well as considerations during thermablation to help inform surgical decision-making.

Concomitant brain abscess and spinal cord abscess in an immunocompetent teenage male: illustrative case.

BACKGROUND: Multiple bilateral brain abscesses occur rarely in immunocompetent patients. Hematogenous spread to the central nervous system (CNS) allows suppuration and abscess formation in the privileged immune environment of the CNS; hematogenous spread to the spinal cord is extremely rare and the combination of multifocal brain abscesses and intramedullary abscesses has not been reported. This report presents a rare presentation and diagrams a treatment algorithm involving iterative minimal access surgeries and prolonged medical management. OBSERVATIONS: The authors present a case of an 18-year-old male with numerous multifocal and bilateral intraparenchymal abscesses and a medically resistant C5 intramedullary spinal cord abscess. The symptomatic patient had a left oculomotor palsy and left hemiparesis, ultimately undergoing ultrasound-guided aspiration of abscesses in the left frontal and left cerebral peduncle. Following transient motor improvement, he evolved tetraparesis prompting spinal cord imaging and emergent ultrasound-guided needle aspiration of an occult C5 intramedullary spinal cord abscess. The patient received appropriate medical therapy, completed inpatient rehabilitation, and made a full recovery. LESSONS: Needle- and ultrasound-guided catheter drainage of CNS abscesses should be considered for symptomatic lesions. Following the neurological examination closely is extremely important; if the expected neurological improvement is delayed or regresses, then expanded imaging is warranted.

Relationship between haematological profile and progression or spontaneous regression of melanoma in the Melanoma-bearing Libechov Minipigs.

Haematological parameters, plasma iron concentration, and bodyweight were monitored in Melanoma-bearing Libechov Minipigs (MeLiM) from 5 to 18 weeks old. Animals with melanoma progression (P group) and spontaneous regression (SR group) were compared. The P group showed the lowest median values of red blood cell counts (RBC), haematocrit (HCT), haemoglobin concentration (HGB), and bodyweight, whereas the control white (tumour-free) pigs (C group) revealed the highest mean values of these parameters. The mean values of pigs with SR fell between the P and C groups. In addition, a stable concentration of plasma iron was found in the C group, while iron deficiency that increases with age was observed in the MeLiM groups. These results indicate that MeLiM are affected by cancer-related microcytic hypochromic anaemia. The lowest values of HGB, RBC, and HCT, together with the highest number of platelets (PLT) in the P group correspond to melanoma progression. Higher values of these parameters and lower PLT in the MeLiM pigs with SR reflected health improvement due to the destruction of melanoma cells during spontaneous regression. Monitoring of these haematological parameters can help distinguish MeLiM piglets with progression and spontaneous regression of melanoma in the early stages of postnatal development. The findings of this study correspond to findings in human patients in which cancer-related anaemia, thrombocytosis, and iron deficiency are often diagnosed.

[Epidemiological significance of the metabolic syndrome]. / Epidemiologický význam metabolického syndromu.

From an epidemiological point of view, the metabolic syndrome is a group of risk factors causally, rather than coincidentally, related to insulin resistance. The metabolic syndrome is a condition with relatively high prevalence rates in both the Czech Republic and in other developed countries. There is a clear trend of increasing prevalence in both sexes depending on age. In the Czech Republic, the syndrome is less common in females (25.5%) than in males (37.6%). Epidemiological studies found white (Europoid race) males to be at higher risk due to abdominal obesity. The definition of the metabolic syndrome has evolved over time and helps to identify individuals at high risk of developing cardiovascular disease and type 2 diabetes, hence the use of the term cardiometabolic syndrome. Early detection of metabolic syndrome symptoms including insulin resistance should be performed mainly by general practitioners as part of regular check-ups.

Identification of differentially expressed genes in spontaneously regressing melanoma using the MeLiM swine model.

Partial and some few cases of complete spontaneous regression have been observed in cutaneous melanoma patients but little is known about the molecular mechanisms involved. The Melanoblastoma-bearing Libechov Minipig (MeLiM) is a suitable animal model to study the phenomenon of spontaneous regression because MeLiM pigs exhibit naturally occurring melanomas which regress completely 6 months after birth. In this study, we used suppression subtractive hybridization (SSH) to identify molecular determinants of melanoma regression within swine melanoma tissues and melanoma cell cultures. Several markers involved in cell-adhesion, -communication, -motility, signal transduction, negative regulation of cell proliferation, transport and immune response were identified that correlated with melanoma regression whereas the main genes involved in melanin synthesis showed a strong downregulation. For the most differentially expressed genes, we validated the results obtained by SSH with qRT-PCR and with immunohistochemistry for some of them (CD9, MITF, RARRES1). Most notable, for the first time in melanoma, we identified the retinoic acid responder 1 gene (RARRES1) as a main actor of the regression process in melanoma. This first gene expression study in swine melanoma regression, may contribute to the finding of new therapeutic targets for human melanoma treatment.

Multiplex analysis of cytokines involved in tumour growth and spontaneous regression in a rat sarcoma model.

The aim of our study was to examine in vivo and in vitro cytokines produced by Lewis ratderived R5-28 sarcoma cells. These cells produce rapidly growing tumours in approximately two weeks after subcutaneous inoculation. However, spontaneous tumour regression was noted in about 40% of animals. For an explanation of this phenomenon, we evaluated the profile of 19 cytokines during tumour growth and spontaneous regression by the use of "antibody array". To detect cytokines directly originated by the sarcoma, the R5-28 cells were cultivated in vitro and then both the supernatants and the cell lysates were analysed. Our experiments showed three cytokines (MCP-1, TIMP-1 and VEGF) to be produced by R5-28 cells in vitro. Moreover, in vivo, another three cytokines (TNF-alpha, beta-NGF and LIX) were detected both in blood sera and tumour lysates, probably produced by immune and stromal cells during tumour growth. Changes in their expression after spontaneous regression are discussed.

Treatment of recalcitrant venous leg ulcers with autologous keratinocytes in fibrin sealant: a multinational randomized controlled clinical trial.

In a multicenter trial, the effect of a commercially available combination of autologous keratinocytes (3-6 x 10(6)/mL) with fibrin sealant (Tissucol Duo S Immuno, Baxter Hyland Immuno) on the healing of recalcitrant venous leg ulcers (duration >3 months) was compared with standard care. The primary endpoint was time to healing, and the secondary endpoint was number of healed ulcers in both groups. Both groups received compression therapy with short-stretch bandages. Forty-four (38.3%) of the 116 patients who had BioSeed-S treatment achieved complete healing of the target ulcer compared with 24 (22.4%) of 109 patients who received standard treatment. The advantage for treatment with BioSeed-S over standard treatment was statistically significant (chi-square test: p=0.0106). Time to complete healing of ulcers: the log-rank test for equality over strata revealed a superiority of treatment with BioSeed-S+compression (median: 176 days) over compression+standard care (median >201 days) (p<0.0001). This study, to date the largest multicenter study with autologous keratinocytes, provides evidence for its efficacy in the treatment of patients with therapy-resistant chronic venous leg ulcers.

Immune characterization of the Lewis rats inoculated with K2 sarcoma cell line and newly derived R5-28 malignant cells.

Sarcoma is a relatively rare malignant disease with high mortality, bad prognosis and response to conventional therapy. Two possible models of this disease were tested: the K2 rat sarcoma cell line, which was described previously, and the new rat R5-28 cell line derived from a spontaneously growing rat neoplasm with sarcoma morphology. While all rats inoculated with K2 cells developed tumours at 22th-25th day after inoculation (D = 22-25), only 60%-75% of R5-28-inoculated rats were affected by tumours. The frequency and progress of the disease depended on the number of inoculated cells. No metastases were detected in both cases. All affected animals showed large splenomegaly. A possible response of some immune system components to tumours was tested. No tumour-infiltrating lymphocytes were revealed in the tumour tissue. Anti-tumour antibodies were not found in tumour-bearing animal sera. Appropriate changes in peripheral blood lymphocyte subsets were explored. While the relative numbers of both NK cells and Tc were impaired, no changes were noted in numbers of CD4+CD8- T helper cells. Leukocytosis with highly increased numbers of CD11b+ myeloid cells displaying variable expression of CD4 was detected in terminal stages of the disease.

Opposite regulation of tenascin-C and tenascin-X in MeLiM swine heritable cutaneous malignant melanoma.

Interactions between tumour cells and surrounding extracellular matrix (ECM) influence the growth of tumour cells and their ability to metastasise. It is thus interesting to compare ECM composition in tumours and healthy tissues. Using the recently described MeLiM miniature pig model of heritable cutaneous malignant melanoma, we studied the expression of two ECM glycoproteins, the tenascin-C (TN-C) and tenascin-X (TN-X), in normal skin and melanoma. Using semiquantitative RT-PCR, we observed a 3.6-fold mean increase of TN-C RNAs in melanoma compared to normal skin. Both stromal and tumour cells synthesise TN-C. On the contrary, TN-X RNAs decreased 30-fold on average in melanoma. This opposite regulation of TN-C and TN-X RNAs was confirmed at the protein level by indirect immunofluorescence. Whereas pig normal skin displayed a discrete TN-C signal at the dermo-epidermal junction, around blood vessels and hair bulbs, the swine tumour showed enhanced expression of TN-C in these areas and around stromal and tumour cells. In contrast, normal skin showed a strong TN-X staining at the dermo-epidermal junction and in the dermis, whereas this signal almost completely disappeared in the tumour. The results presented here describe a dramatic alteration of the ECM composition in swine malignant melanoma which might have a large influence on tumourigenesis or invasion and metastasis of melanoma cells.

Hereditary melanoblastoma in miniature pigs and its successful therapy by devitalization technique.

Applying selective breeding for 10 years we established the MeLiM (Melanoblastoma-bearing Libechov Minipigs) strain. Melanoblastoma (MB) in this strain shows a hereditary occurrence. Cutaneous tumours are usually nodular, multiple and distributed on various parts of body. They appear in darkly pigmented animals already at the birth or during two months thereafter (57% of all animals). Numerous organ metastases mainly into the spleen, lymph nodes and lungs are regularly ascertained in animals with cutaneous MB. Tumour cells were surprisingly found also in the inner organs of phenotypically healthy minipigs in which no cutaneous MBs were observed visually (27% of all animals). About 34% of all affected piglets die during the first 2 months of age. These features document a malignancy of this tumour in the MeLiM strain. Original surgical technique was applied in more than 40 affected minipigs at 1-2 months of age. It consists in a devitalization (ischemization) of one of cutaneous tumours by the mattress sutures conducted around the tumour base without any excision of tumour tissue. This simple procedure causes a total destruction of MB cells in all cutaneous tumours as well as in all organ metastases during 4-6 months. Animals treated by this technique were fully healed of tumour cells and no relapses were observed. This technique could bring similar positive results also in therapy of human MB.

Antimycobacterial activity of 3'- and 4'-fluorothiobenzanilides.

On the basis of a preliminary study of the antimycobacterial activity of thiobenzanilides, a group of 3'-fluoro- and 4'-fluorothiobenzanilides has been synthesized and tested against Mycobacterium tuberculosis, M. kansasii, M. avium and M. fortuitum. The results of this study demonstrate that electron withdrawing groups increase the activity of thiobenzanilides and fluoro benzothioanilides against atypical strains which is higher then that of INH.

Immunocytochemical characterisation of rabbit and mouse embryonic fibroblasts.

By using indirect immunofluorescence we demonstrated the localisation of extracellular matrix (ECM) proteins (laminin--LAM, collagen IV--COL IV, fibronectin--FN) and the basic fibroblast growth factor (bFGF) in rabbit and mouse primary embryonic fibroblasts (PEF). Proliferating mitotically arrested PEF (by mitomycin C) were compared in both species. The stability of protein expression was ascertained during the first five successive passages. In addition, STO cells (i.e. permanent line of irradiated mouse fibroblasts) were similarly analysed. Rabbit PEF showed very high extracellular staining for FN and a negligible cytoplasmic positivity for LAM and COL IV. A totally reversed staining pattern for ECM proteins was found in mouse PEF. A dense cytoplasmic granulation (concentrated around the nucleus) was revealed for LAM and COL IV and almost no reaction for FN. The staining patterns were very stable at the culture conditions we applied. They were maintained during the first five successive passages in proliferating as well as non-proliferating mouse and rabbit PEF and were independent of cell concentration (individually dispersed cells versus cells in a confluent layer). STO cells showed the same staining for ECM proteins as the mouse PEF, thus confirming their origin from the same animal species. Light granular staining for bFGF was found in the cytoplasm of proliferating and mitotically arrested rabbit and mouse PEF and STO cells. The differences in expression of ECM proteins between the rabbit and mouse PEF, as well as the synthesis of bFGF, should be taken into consideration when these cells are used in vitro as a feeder layer for various cells (e.g. embryonic stem cells).

Embryoglycans regulate FGF-2-mediated mesoderm induction in the rabbit embryo.

Several peptide growth factors, including members of the fibroblast growth factor (FGF) superfamily, are potential inducers of mesoderm in vertebrates. Receptor binding of basic FGF (FGF-2) is promoted by cell surface or extracellular matrix proteoglycans. The substantial biosynthesis of proteoglycans by embryonic cells (called embryoglycans) and their potential role as ligands for growth factor receptors led us to examine the role of embryoglycans that carry the developmentally regulated oligosaccharide epitope TEC 1, in the binding of FGF-2 to cultured rabbit inner cell masses (ICMs). Culture of isolated ICMs in the presence of FGF-2 gave rise to well delimited colonies with migrating cells at the periphery. In these cells, TEC 1 staining shifts from a punctate pattern over the entire membrane, to an apical, finely granular distribution with some internalization. This shift occurs after 96 hours in culture. Here we show that: (1) migrating cells are mesoderm-like in phenotype; (2) antibodies against TEC 1 blocked FGF-2 mediated differentiation in vitro; (3) antibodies against TEC 1 selectively blocked binding of FGF-2 to ectodermal receptors and, vice versa, the binding of TEC 1-specific antibodies to ectodermal cells can be competed by excess FGF-2; (4) the same switch in TEC 1 staining patterns was observed in vivo, between the day 7 and the day 9 rabbit embryo. These data suggest the involvement of defined species of embryonic cell surface epitopes in the regulation of FGF-2 receptor binding. Moreover, this proposed binding activity is temporally restricted to ectodermal cells and disappears early during differentiation. Thus, the apical TEC 1 redistribution can be considered as the earliest indicator of mesoderm formation.

Ultrastructural demonstration of glucose-6-phosphatase activity and glycogen in skeletal muscles of newborn piglets with the splayleg syndrome.

The ultrastructural localization of glucose-6-phosphatase activity and glycogen were investigated in the longissimus dorsi and biceps femoris muscles of normal and splaylegged newborn piglets. Significant differences were ascertained in the distribution of the reaction product of glucose-6-phosphatase activity between the two groups of animals. A fine precipitate was found in the sarcoplasmic reticulum and in the perinuclear cisternae of normal piglet muscles. In splaylegged muscles, variable deposits of coarse reaction product were observed within the extremely dilated cisternae of sarcoplasmic reticulum at their periphery. Moreover, both longitudinal and transversal ultrathin sections of these muscles showed a reduced number of myofibrils and an increased accumulation of glycogen (especially within the large extramyofibrillar spaces) in comparison with muscles of normal piglets.

M-twist expression inhibits mouse embryonic stem cell-derived myogenic differentiation in vitro.

The mouse M-twist gene codes for a basic helix-loop-helix protein which was shown to be inhibitory for differentiation of myogenic cells in culture. Mouse embryonic stem (ES) cells of line BLC6 efficiently differentiating into skeletal muscle cells when cultivated as embryo-like aggregates (embryoid bodies) were stably transfected with the plasmid pME18s-twist containing the M-twist gene under the control of the modified SV40 early promoter SR alpha. Two pME18s-twist-expressing clones showed delayed and reduced skeletal muscle cell differentiation depending on the level of exogenous M-twist expression compared to control cells. By morphological analysis using phase contrast microscopy and hematoxylin-eosin staining, the development of first myocytes and formation of myotubes in embryoid body outgrowths of these clones were found to be delayed for about 3 days in comparison to control cells. Immunofluorescence studies with a monoclonal antibody against sarcomeric myosin heavy chain revealed that myogenic cells appeared in so-called myogenic centers showing a reduced number of myocytes and myotubes in the M-twist-expressing clones. Using RT-PCR analysis the expression of the skeletal muscle determination genes myf5, myogenin, and MyoD as well as muscle-specific genes coding for the gamma-subunit of the nicotinic acetylcholine receptor and the cell adhesion molecule M-cadherin were found to appear with a delay of at least 1 to 4 days in the pME18s-twist-transfected cells during the development of embryoid bodies. We conclude that the constitutive expression of the mouse M-twist gene during ES-cell-derived differentiation has an inhibitory effect on skeletal muscle cell development depending on the level of exogenous M-twist expression.