Evaluation of prognostic biomarkers in a population-validated Finnish HNSCC patient cohort.

INTRODUCTION: Prognostic biomarkers and novel therapeutic approaches have been slow to emerge in the treatment of head and neck squamous cell carcinoma (HNSCC). In this study, an HNSCC patient cohort is created and performance of putative prognostic biomarkers investigated in a population-validated setting. The overall goal is to develop a novel way to combine biomarker analyses with population-level clinical data on HNSCC patients and thus to improve the carryover of biomarkers into clinical practice. MATERIALS AND METHODS: To avoid selection biases in retrospective study design, all HNSCC patients were identified and corresponding clinical data were collected from the Southwest Finland geographical area. A particular emphasis was laid on avoiding potential biases in sample selection for immunohistochemical staining analyses. Staining results were evaluated for potential prognostic resolution. RESULTS: After comprehensive evaluation, the patient cohort was found to be representative of the background population in terms of clinical characteristics such as patient age and TNM stage distribution. A negligible drop-out of 1.3% (6/476) was observed during the first follow-up year. By immunohistochemical analysis, the role of previously implicated HNSCC biomarkers (p53, EGFR, p16, CIP2A, Oct4, MET, and NDFIP1) was investigated. DISCUSSION: Our exceptionally representative patient material supports the use of population validation to improve the applicability of results to real-life situations. The failure of the putative prognostic biomarkers emphasizes the need for controlling bias in retrospective studies, especially in the heterogenous tumor environment of HNSCC. The resolution of simple prognostic examination is unlikely to be sufficient to identify biomarkers for clinical practice of HNSCC.

Toll-like receptor 5 and 7 expression in adenoid cystic carcinoma of major salivary glands.

Adenoid cystic carcinoma (ACC) of the salivary glands has a poor long-term prognosis and high metastatic rate. Toll-like receptors (TLRs) have been related to tumour progression but have also tumour growth-inhibiting responses. To the best of our knowledge, they have not been studied previously in ACC. We studied the immunoexpression of TLR 5 and 7 in ACC of the major salivary glands. From a cohort of 54 patients with ACC of the major salivary glands treated at the Department of Otorhinolaryngology-Head and Neck Surgery, Helsinki University Hospital, Helsinki, Finland in 1974-2009, there were 34 primary tumours and six metastases available for immunohistochemical analysis. Immunohistochemical expression of TLR 5 and 7 were correlated to clinicopathological findings and patient survival. Both TLR 5 and 7 were expressed in ACCs and their metastases, mostly on the cell membranes. The expression was heterogeneous in individual tumours. TLR 5 was expressed less in male samples, and TLR 7 had lower expression in ACCs with solid growth pattern. No correlation with survival was found. In the normal salivary gland, the TLR 5 and 7 expression was mainly negative. Both TLR 5 and 7 are expressed in salivary adenoid cystic carcinoma on the cell membranes as well as in cytoplasm.

Bmi-1 expression predicts prognosis in squamous cell carcinoma of the tongue.

BACKGROUND: The prognosis of squamous cell carcinoma of the oral tongue is poor and it would be beneficial to find prognostic markers to better adjust treatment. Bmi-1 controls cell cycle and self-renewal of tissue stem cells, transcription factor c-myc affects cell proliferation and apoptosis, and Snail regulates epithelial-mesenchymal transition. The expression of these markers has been connected to prognosis in many cancer types. METHODS: Bmi-1, c-myc, and Snail expressions were studied in our material consisting of 73 primarily T1N0M0 oral tongue carcinoma patients. We compared the immunoexpressions of Bmi-1, c-myc, and Snail with clinical parameters including the degree of histological differentiation, tumour size, TNM classification, depth of invasion, and resection margins. In addition, survival analyses were performed, comparing disease-free survival time with the registered protein expression of the markers mentioned above. RESULTS: A significant correlation between Bmi-1 protein expression and recurrence (log-rank test, P=0.005) was detected. Snail and c-myc expression did not correlate with prognosis. Snail expression correlated with histopathological grade (Fisher's exact test, P=0.007) and with the invasion depth of tumours (chi(2)-test, P=0.037). CONCLUSION: Negative Bmi-1 immunoexpression might serve as a marker of poor prognosis in oral tongue carcinoma patients.

C3d fragment of complement interacts with laminin and binds to basement membranes of glomerulus and trophoblast.

Two mouse monoclonal antibodies generated against human placental homogenate were found to react specifically with human complement component C3. In immunofluorescence of human tissues, these antibodies gave a bright linear staining outlining the glomerular basement membrane of the adult kidney and the trophoblast basement membrane of placenta. An identical staining pattern was observed with a rabbit C3d antiserum which also prevented binding of the monoclonal antibodies to tissue sections. Only negligible basement membrane staining was observed in the same tissues with antisera to human C3c, C5, IgG, IgA, or IgM. When interactions of C3 with basement membrane proteins were tested in enzyme immunoassays and column chromatography, C3(H2O) was found to bind efficiently to solid-phase laminin. Native C3 from fresh plasma did not bind to laminin but C3 from plasma treated with methylamine bound efficiently. When C3 was cleaved with trypsin, C3b and C3d but not C3c bound to laminin-Sepharose. The interaction of C3 and laminin was inhibited by soluble laminin and by high ionic strength. The results indicate that C3d, a biologically active breakdown product of C3, can be found in glomerular and placental basement membranes in the absence of signs for ongoing local complement activation or immune complex deposition. It is possible that binding affinities between C3 and basement membrane molecules, especially laminin, are involved in the retention of C3d at these sites. Such interactions between C3 and components of the glomerular basement membrane could play important roles in complement-related pathological processes of the glomerulus.

Identification of target genes in laryngeal squamous cell carcinoma by high-resolution copy number and gene expression microarray analyses.

Molecular mechanisms contributing to initiation and progression of head and neck squamous cell carcinoma are still poorly known. Numerous genetic alterations have been described, but molecular consequences of such alterations in most cases remain unclear. Here, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 20 laryngeal cancer cell lines and primary tumors. Our aim was to identify genetic alterations that play a key role in disease pathogenesis and pinpoint genes whose expression is directly impacted by these events. Integration of DNA level data from array-based comparative genomic hybridization with RNA level information from oligonucleotide microarrays was achieved with custom-developed bioinformatic methods. High-level amplifications had a clear impact on gene expression. Across the genome, overexpression of 739 genes could be attributed to gene amplification events in cell lines, with 325 genes showing the same phenomenon in primary tumors including FADD and PPFIA1 at 11q13. The analysis of gene ontology and pathway distributions further pinpointed genes that may identify potential targets of therapeutic intervention. Our data highlight genes that may be critically important to laryngeal cancer progression and offer potential therapeutic targets.

Genetic changes in sporadic keratocystic odontogenic tumors (odontogenic keratocysts).

Little is known about the genetic background of keratocystic odontogenic tumors (KCOT, odontogenic keratocysts). Our aim was to characterize genomic aberrations in sporadic KCOT using cDNA-expression arrays and array-comparative genomic hybridization. For cDNA-expression arrays, 10 KCOT specimens and 20 fetal tooth germs were studied. Quantitative real-time reverse-transcription/polymerase chain-reaction and immunohistochemical studies were also undertaken. Several genes were over-expressed in 12q13, including cytokeratin 6B (KRT6B) ( approximately 10-fold), epidermal growth factor receptor ERBB3 (approximately 4.7-fold), and glioma-associated oncogene homologue 1 (GLI1) (approximately 5- to 12-fold). One amplicon (approximately 0.7 Mega base pairs [Mbp]), covering several genes involved in the regulation of cell growth, was found in 12q13.2. Deletions were found in 3q13.1, 5p14.3, and 7q31.3, including the cell-adhesion-related gene cadherin 18 (CDH18) and leukocyte cell adhesion molecule (ALCAM, MEMD). Over-expressed and amplified genes in 12q13, also reported in several other tumors and cell lines, may contribute to the persistent growth characteristics of KCOT.

The MDM2 promoter polymorphism SNP309T-->G and the risk of uterine leiomyosarcoma, colorectal cancer, and squamous cell carcinoma of the head and neck.

BACKGROUND: MDM2 acts as a principal regulator of the tumour suppressor p53 by targeting its destruction through the ubiquitin pathway. A polymorphism in the MDM2 promoter (SNP309) was recently identified. SNP309 was shown to result, via Sp1, in higher levels of MDM2 RNA and protein, and subsequent attenuation of the p53 pathway. Furthermore, SNP309 was proposed to be associated with accelerated soft tissue sarcoma formation in both hereditary (Li-Fraumeni) and sporadic cases in humans. METHODS: We evaluated the possible contribution of SNP309 to three tumour types known to be linked with the MDM2/p53 pathway, using genomic sequencing or restriction fragment length polymorphism as screening methods. Three separate Finnish tumour materials (population based sets of 68 patients with early onset uterine leiomyosarcomas and 1042 patients with colorectal cancer, and a series of 162 patients with squamous cell carcinoma of the head and neck) and a set of 185 healthy Finnish controls were analysed for SNP309. RESULTS: Frequencies of SNP309 were similar in all four cohorts. In the colorectal cancer series, SNP309 was somewhat more frequent in women and in patients with microsatellite stable tumours. Female SNP309 carriers were diagnosed with colorectal cancer approximately 2.7 years earlier than those carrying the wild type gene. However, no statistically significant association of SNP309 with patients' age at disease onset or to any other clinicopathological parameter was found in these three tumour materials. CONCLUSION: SNP309 had no significant contribution to tumour formation in our materials. Possible associations of SNP309 with microsatellite stable colorectal cancer and with earlier disease onset in female carriers need to be examined in subsequent studies.

Bone morphogenetic protein-6 is a marker of serous acinar cell differentiation in normal and neoplastic human salivary gland.

Bone morphogenetic protein (BMP-6, also known as vegetal-pale-gene-related and decaplentaplegic-vegetal-related) is a member of the transforming growth factor-beta superfamily of multifunctional signaling molecules. BMP-6 appears to play various biological roles in developing tissues, including regulation of epithelial differentiation. To study the possible involvement of BMP-6 in normal and neoplastic human salivary glands, we compared its mRNA and protein expression in 4 fetal and 15 adult salivary glands and in 22 benign and 32 malignant salivary gland tumors. In situ hybridization and Northern blot analysis indicated that BMP-6 transcripts are expressed at low levels in acinar cells of adult submandibular glands but not in ductal or stromal cells. BMP-6 was immunolocated specifically in serous acini of parotid and submandibular glands. None was found in primitive fetal acini or any other types of cell in adult salivary glands, including mucous acini and epithelial cells of intercalated, striated, and excretory ducts. All 16 cases of acinic cell carcinoma consistently exhibited cytoplasmic BMP-6 staining in the acinar tumor cells. Other cell types in these tumors, including intercalated duct-like cells, clear, vacuolated cells, and nonspecific glandular cells, exhibited no cytoplasmic BMP-6 staining. Other benign and malignant salivary gland tumors lacked BMP-6 immunoreactivity, except in areas of squamous differentiation. The results indicate that in salivary glands, BMP-6 expression is uniquely associated with acinar cell differentiation and suggest that BMP-6 may play a role in salivary gland function. More importantly, our experience of differential diagnostic problems related to salivary gland tumors suggests that the demonstration of consistent and specific BMP-6 immunoreactivity in acinic cell carcinoma is likely to be of clinical value.

Clinicopathological characteristics and cell cycle proteins as potential prognostic factors in myoepithelial carcinoma of salivary glands.

Myoepithelial carcinoma (MCA) is a rare malignancy of salivary glands that was included in the WHO Classification of Head and Neck Tumors in 1991. MCA has shown a broad spectrum of clinical outcomes, but attempts to identify prognostic markers for this malignancy have not resulted in significant progress. Conventional histopathological characteristics such as tumour grade, nuclear atypia, mitotic index and cell proliferation have failed to predict the outcome of MCA. In this study, we reviewed the histopathology of 19 cases of MCA focusing on nuclear atypia, mitotic count, tumour necrosis, nerve and vascular invasion and occurrence of a pre-existing pleomorphic adenoma in connection to the MCA. Histopathological characteristics and clinical information were correlated with the immunohistochemical expression of cell cycle proteins including c-Myc, p21, Cdk4 and Cyclin D3. The proportion of tumour cells immunoreactive for these markers and their intensity of staining were correlated with clinical information using logistic regression, Kaplan-Meier and Cox regression. Using logistic regression analysis, cytoplasmic c-Myc expression was associated with the occurrence of metastases (P = 0.019), but limitations of semi-quantitation of immunostaining and the limited number of cases preclude definitive conclusions. Our data show that the occurrence of tumour necrosis predicts poor disease-free survival in MCA (P = 0.035).

Basement membrane matrices in mouse embryogenesis, teratocarcinoma differentiation and in neuromuscular maturation.

In this paper we discuss studies on basement membrane and interstitial matrix molecules in early development and teratocarcinoma differentiation. In the early embryo a compartmentalization of newly formed cell types takes place immediately by formation of basement membranes. The stage-specific developmental appearance of extracellular matrix molecules such as type IV collagen, laminin, entactin, fibronectin and proteoglycans seems to reflect a diversified role of extracellular matrices already in the earliest stages of development. In teratocarcinoma cultures the appearance and composition of extracellular matrices during the differentiation of endoderm cells closely resembles that found in the early embryo. Also in this respect the teratocarcinoma system can be used as a model for studies on early development. In later developmental phenomena other matrix molecules can also be of importance. Merosin, a novel tissue-specific basement membrane-associated protein that appears during muscle and nerve maturation is an example of such molecules.

Early dental epithelial transcription factors distinguish ameloblastoma from keratocystic odontogenic tumor.

The aim of the study was to characterize the molecular relationship between ameloblastoma and keratocystic odontogenic tumor (KCOT) by means of a genome-wide expression analysis. Total RNA from 27 fresh tumor samples of 15 solid/multicystic intraosseous ameloblastomas and 12 sporadic KCOTs was hybridized on Affymetrix whole genome arrays. Hierarchical clustering separated ameloblastomas and KCOTs into 2 distinct groups. The gene set enrichment analysis based on 303 dental genes showed a similar separation of ameloblastomas and KCOTs. Early dental epithelial markers PITX2, MSX2, DLX2, RUNX1, and ISL1 were differentially overexpressed in ameloblastoma, indicating its dental identity. Also, PTHLH, a hormone involved in tooth eruption and invasive growth, was one of the most differentially upregulated genes in ameloblastoma. The most differentially overexpressed genes in KCOT were squamous epithelial differentiation markers SPRR1A, KRTDAP, and KRT4, as well as DSG1, a component of desmosomal cell-cell junctions. Additonally, the epithelial stem cell marker SOX2 was significantly upregulated in KCOT when compared with ameloblastoma. Taken together, the gene expression profile of ameloblastoma reflects differentiation from dental lamina toward the cap/bell stage of tooth development, as indicated by dental epithelium-specific transcription factors. In contrast, gene expression of KCOT indicates differentiation toward keratinocytes.

A simple novel prognostic model for early stage oral tongue cancer.

The prognostication of patient outcome is one of the greatest challenges in the management of early stage oral tongue squamous cell carcinoma (OTSCC). This study introduces a simple histopathological model for the prognostication of survival in patients with early OTSCC. A total of 311 cases (from Finland and Brazil) with clinically evaluated early stage OTSCC (cT1-T2cN0cM0) were included in this multicentre retrospective study. Tumour budding (B) and depth of invasion (D) were scored on haematoxylin-eosin-stained cancer slides. The cut-off point for tumour budding was set at 5 buds (low <5; high ≥5) and for depth of invasion at 4mm (low <4mm; high ≥4mm). The scores of B and D were combined into one model: the BD predictive model. On multivariate analysis, a high risk score (BD score 2) correlated significantly with loco-regional recurrence (P=0.033) and death due to OTSCC (P<0.001) in early stage OTSCC. The new BD model is a promising prognostic tool to identify those patients with aggressive cases of early stage OTSCC who might benefit from multimodality treatment.

Localization of progesterone-associated endometrial protein mRNA by in-situ hybridization in human pregnancy decidua, endometriosis and borderline endometrioid adenoma.

Progesterone-associated endometrial protein (PAEP) has been isolated from human decidualized endometrium. In-situ hybridization histochemistry was employed to determine the cellular localization of PAEP mRNA in decidua during pregnancy. PAEP mRNA was found to be expressed in the glandular epithelium of decidua spongiosa throughout pregnancy. Substantial variations in the amount of PAEP mRNA during the course of pregnancy were observed, and it was most abundant at the end of the first trimester. We also found that the PAEP gene was expressed in endometriosis and in a borderline endometrioid adenoma. As in decidual tissues, PAEP mRNA in endometriosis was abundant in the glandular compartment.

Muscle membrane-skeleton protein changes and histopathological characterization of muscle-eye-brain disease.

Muscle-eye-brain disease belongs to congenital muscular dystrophies with central nervous system abnormalities. The etiology of MEB is still unknown, but abnormal immunoreactivity for laminin-2 has been reported. To evaluate disease progression in muscle tissue, 32 biopsy specimens from 17 muscle-eye-brain patients were analysed. The samples of four patients were studied by immunohistochemical techniques and by quantitative Western blotting. The samples showed a great variation in the muscle pathology. Regenerative fibers and mild fiber size variation were present in over 60%. At infancy, necrotic and regenerative fibers were common, while fat infiltration was the most prominent finding in the age group over five years. In quantitative studies, the amount of laminin alpha 2 chain was clearly reduced to 10-20% of normal. In contrast, laminin beta 2 chain was overexpressed in the Western blotting studies. These findings may reflect a yet unidentified primary disturbance in the basement membrane composition and function.

Basement membrane-like matrix of teratocarcinoma-derived endodermal cells: presence of laminin and heparan sulfate in the matrix at points of attachment to cells.

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.

Anchoring complex components laminin-5 and type VII collagen in intestine: association with migrating and differentiating enterocytes.

Anchoring complex component laminin-5 and its subunits laminin (Ln)-alpha3 and Ln-beta3 chains, Type VII collagen, and integrin chains alpha3, alpha6, and beta4 were studied in developing and adult human intestine and compared with findings on Ln-alpha1 and Ln-alpha2 chains. In adult human duodenum, jejunum, and ileum, Ln-5 detected with a polyclonal antiserum and Ln-alpha3 and Ln-beta3 chains, detected with monoclonal antibodies (MAbs), were restricted to the epithelial basement membranes (BMs) of villi, whereas Ln-alpha2 chain was seen only focally in crypt bottoms. In double labeling experiments, the stretch of crypt BM corresponding to the proliferative cell compartment was found to be devoid of both Ln-alpha3 and Ln-alpha2 chains. Double labeling for Ln-5 and proliferating cell nuclear antigen also showed an abrupt onset of Ln-5 expression exactly at the upper edge of the proliferative cell compartment. Type VII collagen was negligible in duodenum and showed a rising duodenal-ileal gradient localizing to villar BMs. Double labeling for Ln-5 and Type VII collagen, however, indicated only partial co-distribution in the intestine. Electron microscopy of ileum revealed both anchoring filaments and anchoring fibrils but no hemidesmosomal plaques. Our results demonstrate the expression of Ln-5 in BMs outside of stratified epithelia and indicate that Ln-5 in the intestine is associated with the compartment of migrating and differentiating enterocytes. Absence of hemidesmosomes and the presence of other anchoring complex components, such as Ln-5, Type VII collagen, and integrin chains alpha3, alpha6, and beta4, suggests unique properties for epithelial cell attachment in the intestine.

Analysis of collagen isotypes in crystalloid structures of salivary gland tumors.

Extracellular collagenous crystalloids (CCs) have been reported in salivary gland tumors. To study the occurrence and characteristics of these structures we reviewed 230 pleomorphic adenomas and myoepitheliomas of both major and minor salivary glands. Twelve of these cases contained crystalloids composed of radially arranged collagen fibers. However, no CCs were found in 124 malignant salivary gland tumors of different types. We show that CCs contain types I and III collagen but not type II, IV, or VI collagen. Moreover, cells surrounding CCs expressed the basement membrane molecules laminin and type IV collagen. These cells also showed other immunohistochemical features typical of myoepithelial cells.

Prognostic significance of cell proliferation in mucoepidermoid carcinomas of the salivary gland: clinicopathological study using MIB 1 antibody in paraffin sections.

Mucoepidermoid carcinomas of salivary gland origin have an uncertain clinical course not directly predictable by histomorphology. The MIB 1 antibody, which detects Ki-67 antigen in formalin-fixed, paraffin-embedded tissues, was used to study cell proliferation in these tumors. An MIB 1 index was developed to express the percentage of MIB 1-positive proliferating cells, and the results were compared with histomorphological tumor grade and clinical outcome. All patients with MIB 1 indices lower than 10% in their primary tumors had a favorable clinical outcome. Most patients with MIB 1 indices higher than 10% developed a recurrent or metastasizing disease. All patients who died of their tumor or who had persistent tumor had MIB 1 indices higher than 10%. Thus, the MIB 1 index defines two virtually nonoverlapping forms of the disease, an indolent one and an aggressive one. Cell proliferation in mucoepidermoid carcinomas, assessed with the MIB 1 antibody, thus represents a significant prognostic factor for improving the accuracy of conventional histological grading.

Well-differentiated acinic cell carcinoma of salivary glands associated with lymphoid stroma.

In a multicenter study, 69 acinic cell carcinomas of the salivary glands were identified, of which 12 constituted what the authors believe to be a distinct subgroup. Their most noticeable feature was a dense lymphoid stroma with well-developed germinal centers, surrounding a sometimes scanty epithelial component, which in each case had a microcystic growth pattern. All these tumors were enveloped by a thin fibrous pseudocapsule, thus mimicking an intraparotid lymph node containing a metastasis. All 12 cases showed low MIB1 proliferative activity, with a mean index of 1.7% (range, 0.5 to 3.7). All patients remained well without recurrence or metastasis in followup periods of 19 months to 14 years. A second subgroup of nine acinic cell carcinomas also possessed a heavy lymphoid stroma with germinal centers, but its distribution was more patchy than in the first subgroup, and in addition, the fibrous pseudo-capsule was incomplete or absent. In each case the epithelial growth pattern was other than microcystic. These tumors had significantly higher MIB1 indices (mean, 17%; range, 3.4 to 45). In contrast to the first subgroup, only three of nine patients remained well with no further disease. The other six patients developed recurrences or metastases, and two died of disseminated cancer. In view of the clinical and pathological data, it is speculated that the tumor foci lacking lymphoid stroma in each of the second subgroup possibly represented a clone of high-grade malignancy arising within a low-grade acinic cell carcinoma with lymphoid stroma.

Abnormal mitochondria and sarcoplasmic changes in rabbit skeletal muscle induced by immobilization.

Immobilization of the rabbit knee in extended position results in damage to the vastus intermedius profundus (VIP) muscle. To examine the mechanisms involved in initiation of the injury, we studied the light and electron microscopic morphology of the VIP muscle, as well as the activity and distribution of NADH tetrazolium reductase (NADH-TR) in the affected muscle, and determined serum total creatine kinase (CK) activity in immobilized rabbits. The VIP muscle of the immobilized right hindlimb was removed at various time points (10 h, 24 h, 36 h and 48-72 h, n=5 for each time point). The nonimmobilized left hindlimb and five nonimmobilized animals served as controls. No morphological changes were observed by light microscopy within 48-72 h in routine stainings. Transient ultrastructural abnormalities, including abnormal cristae, matrix lucencies and mild swelling of mitochondria, were observed between 10 h and 36 h of immobilization, subsiding by 48-72 h. On the other hand, progressive disorganization of myofibrils with breaking-up of Z-bands and an increase in the number and size of sarcoplasmic lipid vacuoles was seen with increasing duration of immobilization. NADH-TR activity at subsarcolemmal locations had decreased by 10 h and disappeared by 24 h of immobilization, while the intermyofibrillar mitochondria remained unaltered. Serum total CK activity began to increase by 2 h of immobilization and reached a peak by 24 h. The results indicate that already a few hours of immobilization of the rabbit knee in extension leads to signs of metabolic disturbance of the VIP muscle and sarcolemmal leakage. The simultaneous occurrence of transient mitochondrial abnormalities, transient CK efflux and progressive myofibrillar damage suggests the operation of multiple adverse mechanisms already at the onset of disuse muscle atrophy.