Biodegradable composite scaffolds incorporating an intramedullary rod and delivering bone morphogenetic protein-2 for stabilization and bone regeneration in segmental long bone defects.

In this study, a two-part bone tissue engineering scaffold was investigated. The scaffold consists of a solid poly(propylene fumarate) (PPF) intramedullary rod for mechanical support surrounded by a porous PPF sleeve for osseointegration and delivery of poly(dl-lactic-co-glycolic acid) (PLGA) microspheres with adsorbed recombinant human bone morphogenetic protein-2 (rhBMP-2). Scaffolds were implanted into critical size rat segmental femoral defects with internal fixation for 12 weeks. Bone formation was assessed throughout the study via radiography, and following euthanasia, via microcomputed tomography and histology. Mechanical stabilization was evaluated further via torsional testing. Experimental implant groups included the PPF rod alone and the rod with a porous PPF sleeve containing PLGA microspheres with 0, 2 or 8 µg of rhBMP-2 adsorbed onto their surface. Results showed that presence of the scaffold increased mechanical stabilization of the defect, as evidenced by the increased torsional stiffness of the femurs by the presence of a rod compared to the empty defect. Although the presence of a rod decreased bone formation, the presence of a sleeve combined with a low or high dose of rhBMP-2 increased the torsional stiffness to 2.06 ± 0.63 and 1.68 ± 0.56 N·mm, respectively, from 0.56 ± 0.24 N·mm for the rod alone. The results indicate that, while scaffolds may provide structural support to regenerating tissues and increase their mechanical properties, the presence of scaffolds within defects may hinder overall bone formation if they interfere with cellular processes.

Changes in matrix metalloproteinases and their endogenous inhibitors during tumor progression in the uterine cervix.

PURPOSE: To study the role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cervical tumorigenesis, we analyzed 70 cervical tissue specimens that included 15 low-grade squamous intraepithelial lesions (SILs), 20 high-grade SILs, 25 squamous cell carcinomas (SCCs) and 10 specimens of normal cervical tissue. METHODS: The gelatinolytic activity of MMP-9 and MMP-2 was determined by zymographic analysis. The expression of MMP-9 and MMP-2 and TIMP-1 and TIMP-2 was determined by immunohistochemistry. RESULTS: All the samples had 72/66 kDa gelatinase activity; 92 kDa gelatinase activity was detected only in high-grade SILs and SCCs. Immunohistochemical analysis showed weak positivity for MMP-2 in normal cervical epithelium and low-grade SILs. However, high-grade SILs and SCCs showed intense cellular and stromal reactivity for MMP-2 and MMP-9. For TIMP-1 and TIMP-2, normal cervical epithelium and low-grade SILs showed intense immunostaining, >50% of high-grade SILs showed positivity, and 95% of SCCs showed intense stromal and cellular reactivity. CONCLUSIONS: Increase in the relative activity of these gelatinases and enhanced immunostaining for MMPs and TIMPs with tumor progression suggest that they may play a crucial role in cervical cancer progression. A significant association between stage of the lesion and expression of MMPs and TIMPs ( P<0.01) was found. Immunohistochemical studies indicate that these MMPs may be of basal cell origin in cervical tissue, although the mechanism of their upregulation is not clearly understood.

In situ apoptosis in the thyroid.

Recent evidence has emphasized the importance of programmed cell death, or apoptosis, in the maintenance of tissue homeostasis and pathogenesis of tumors. This study analyzed the significance of apoptosis in relation to the expression of p53 and bcl-2 proteins, tissue proliferation defined by Ki-67 expression, and tissue histology in thyroid tissue. Extent of apoptosis was defined by morphological criteria and the terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate (dUTP) biotin nick end labeling (TUNEL) assay. Immunocytochemistry was performed for p53, bcl-2, and Ki-67 expression. There was good correlation between TUNEL-reactive cells and morphological evaluation criteria for apoptosis. The extent of apoptosis was significantly associated with the type of thyroid lesion (r = 0.66990, p = 0.000012), both proliferative (namely multinodular goiter) and neoplastic (benign and malignant). A higher extent of apoptosis was evident in medullary and anaplastic carcinomas. Apoptosis also correlated to p53 protein accumulation (r = 0.485, p = 0.00041) and Ki-67 immunoreactivity (r = 0.435, p = 0.001). An inverse correlation was observed between bcl-2 expression and the extent of apoptosis (r = -0.33369, p = 0.01912). A direct correlation was also observed between p53 expression and Ki-67 immunoreactivity (r = 0.623, p = 0.0002). By inhibiting apoptosis, bcl-2, may cause a shift in tissue kinetics toward the preservation of genetically aberrant cells, thereby facilitating tumor progression. These results imply that rapidly proliferating tumors appear to have a high cell turnover state in which there may be increased chance of apoptosis among the proliferating cells. The ability of apoptosis to occur in the presence of a possibly mutant p53 protein suggest the existence of at least two p53 dependent apoptotic pathways, one requiring activation of specific target genes and the other independent of it. However, keeping in mind the limited number of subjects studied in each subgroup and the rather low correlation coefficients, these possibilities would have to be substantiated in a larger study population.

Increased angiogenesis in the uterine cervix associated with human papillomavirus infection.

We studied the relationship between angiogenesis (using the CD34 antibody), the presence of human papilloma virus (HPV) infection, HPV E6 protein expression and the accumulation of p53 protein at various phases of tumour progression in the uterine cervix. Expression of CD34, p53 and HPV E6 protein was evaluated by immunocytochemistry. Presence of the mutant p53 was detected using a mutant specific ELISA, and the type of HPV was determined by the Polymerase Chain Reaction. A total of 230 cervical tissue samples were analyzed and included 40 cases of apparently normal cervical epithelium, 37 low grade squamous intraepithelial lesions (SILs), 43 high grade SILs, 36 well-differentiated squamous cell carcinomas (DSCC), 31 moderately differentiated (MDSCC) and 43 poorly differentiated carcinomas (PDSCC). There was an excellent correlation between the extent of angiogenesis and histological abnormality (r = 0.912, p = 0.000004). The least extent of angiogenesis was seen in normal cervical tissue and low grade SILs where the mean (low power) intra lesional vascular density (ILVD) was 12 +/- 1.13 and 25.66 +/- 5.20, respectively. In high grade squamous intraepithelial lesions (SILs), the mean ILVD value was 80.84 +/- 25.57. In well-differentiated squamous cell carcinomas (WDSCC's) the mean value was 144.22 +/- 28.67 while in moderately differentiated squamous cell carcinomas (MDSCC's) the mean value was 166.29 +/- 34.95 and in poorly differentiated tumours (PDSCC's) 192.42 +/- 27.98. The extent of angiogenesis also correlated to presence of HPV (r = 0.505, p = 0.00001). Increased CD34 expression was associated with the presence of HPV types 16 and 18. A similar correlation was also evident in HPV, 16/18 infected cases expressing the E6 protein (r = 0.612, p = 0.000001). CD34 expression also correlated well with p53 accumulation (r = 0.859, p = 0.000002). Presence of HPV infection significantly correlated with the extent of histological abnormality (r = 0.467, p = 0.00001). Expression of E6 also showed this significant correlation (r = 0.644, p = 0.00002). Accumulation of p53 was significantly more elevated in HPV 16-infected lesions (r = 0.518, p = 0.00001) and E6-expressing cells (r = 0.650, p = 0.000004). Only 12 of the 230 cases analyzed showed presence of the mutant p53 protein. Angiogenesis appears to increase with histological abnormality in the uterine cervix. Angiogenesis also appears to be influenced by high risk HPV infection, the expression of the E6 transforming protein and the p53 tumour suppressor protein.

ras and c-myc oncoproteins during tumor progression in the uterine cervix.

AIMS AND BACKGROUND: Altered oncogenic activity is a feature associated with many malignant and premalignant conditions. Among the many oncogenes, ras and myc are commonly altered in many tumors. This study aims to evaluate the expression of ras and c-myc oncoproteins in a total of 204 cervical tissue samples, including premalignant and malignant lesions as well as apparently normal cervical tissue. METHODS AND STUDY DESIGN: Mouse monoclonal antibodies against the three mammalian ras gene products (c-H-ras, c-K-ras, c-N-ras) and the c-myc protein were used to evaluate oncoprotein expression by immunocytochemistry. RESULTS: None of the samples analyzed displayed immunoreactivity for H-ras and K-ras. Normal cervical epithelium showed minimal immunoreactivity for N-ras with about 33% of the samples expressing the protein. More conspicuous expression in normal tissue was displayed by c-myc, with about 90% of the samples expressing the protein (mean value of cells positive=34%). The immunoreactivity for N-ras increased with increasing histological abnormality from low-grade squamous intraepithelial lesions (SIL) to invasive carcinoma. Increased immunoreactivity for N-ras was evident in the basaloid cells of malignant lesions, with the maximum value of 66% found in poorly differentiated squamous cell carcinoma (PDSCC). The percentage of nuclei positive for c-myc also showed a gradual increase from low-grade SIL onwards, the highest positivity being found in PDSCC, where the mean value was 85%. Statistical analysis revealed a good correlation between the expression of N-ras (r=0.8922, P=0.001) and c-myc (r=0.8856, P=0.001) and various histological stages of tumor progression in the cervical epithelium. CONCLUSIONS: These results therefore suggest that c-myc and N-ras oncoproteins are important during tumor progression in the uterine cervix.

High-risk human papillomavirus infection and E6 protein expression in lesions of the uterine cervix.

Pathologic and epidemiologic investigations carried out over the past several years have provided evidence that carcinogenesis in the uterine cervix is a multi-step process involving discreet preinvasive stages. Molecular epidemiologic data also indicate that human papillomavirus (HPV) infection is a critical factor in the tumor progression process. In vitro studies have shown that for the initiation and maintenance of the malignant phenotype, the expression of the HPV-transforming protein E6 is required. The E6 protein produced by the high-risk HPV types 16 and 18 can bind to and inactivate the tumor suppressor protein p53 leading to deregulated proliferation and defective apoptosis, thus facilitating tumor progression. Therefore, determination of the HPV genotype alone may not be sufficient in assessing tumor progression in the uterine cervix. In the present study, a total of 623 cervical tissue samples at various phases of tumor progression were assessed for HPV infection by nonisotopic in situ hybridization (NISH) and for HPV 16/18 E6 protein expression by immunocytochemistry. There was significant correlation between the extent of histological abnormality and HPV infection. Significant correlation (r = 0.707, p = 0.000) was observed between the presence of HPV 16 and high-grade squamous intraepithelial lesions (SILs) and invasive cancer. The odds ratio of a cervical tissue infected with HPV 16 falling into these two categories was 44.57 (95% CI: 27.10, 73.30). The E6 protein also was mostly detected in high-grade SILs and cervical cancer tissue expressing either HPV 16 or 18. It was less frequent in low-grade SILs infected with HPV 16/18 and was absent in benign cervical tissue infected with HPV 16. The odds ratio of an HPV-16/18-infected cervical tissue positive for E6 being a high-grade SIL or invasive cancer was 16.20 (95% CI: 6.06, 43.33). These results thus show the clinical utility of HPV characterization along with the analysis of the transforming protein E6 in the assessment of tumor progression in the uterine cervix.

Photochemically induced fluorimetric detection of tianeptine and some of its metabolites. Application to pharmaceutical preparation.

The photochemically induced fluorescence (PIF) properties of tianeptine and some of its metabolites were investigated in acidic (pH 2.3) water-alcohol mixtures at room temperature. Two PIF methods were developed, including bulk solution and flow injection analysis (FIA). Linear calibration plots were established over a concentration range of more than one order of magnitude. Limits of detection ranged from 15 ng ml-1 for FIA-PIF to 25 ng ml-1 in bulk solution. The RSDs were between 3 and 5%. The PIF methods were applied to the determination of tianeptine in a pharmaceutical preparation with recoveries varying from 96 to 106% in bulk solutions and from 98 to 106% for FIA-PIF.

Cytokeratins and the evaluation of tumor differentiation in squamous lesions of the uterine cervix.

The differential expression of cytokeratins in epithelial or squamous cells has been demonstrated to be altered during the process of carcinogenesis. This altered expression of cytokeratins (CKs) may be closely related with epithelial differentiation and may remain stable in malignant tumors. In the present study an analysis using two monoclonal antibodies, CK 8.12 antibody specific for CK type 13 and 16 and CK 8.60 antibody specific for CK type 1, 10 and 11 was done in different grades of lesions in the uterine cervix. Changes from the normal expression pattern were seen in high grade Squamous intraepithelial lesions (SIL) (CIN-2/3) and invasive squamous cell carcinoma (SCC). No conspicuous difference in the staining expression between normal/benign cervical tissue and low grade SIL (CIN-I) was evident. Statistical analysis also revealed a significant correlation between the expression of these CK types to the differentiation status of the cervical lesions analyzed. Alterations in the expression of these CKs can be correlated to the differentiation pathway which may be deregulated during cervical carcinogenesis. The findings of the present study suggest that the expression of CK types 13 and 16 and 1, 10 and 11 using CK 8.12 and CK 8.60 antibodies respectively may serve as markers of differentiation in cervical squamous neoplasms.

p53 protein and tumorigenesis in the uterine cervix.

The relationship between molecular abnormalities of p53 tumor suppressor gene product and cancer has been well documented. That correlation may exist between immunocytochemically detectable amount of p53 protein and neoplasia is evidenced by several studies. Detection of p53 protein by immunocytochemistry varies depending on the methods and antibodies used. It has been suggested that the quantitative aspect of p53 protein expression and the proportion of cells expressing p53 may be of clinical importance in human malignancies. In the present study, we have examined the expression of p53 protein in various grades of lesions of the uterine cervix. Statistical analysis showed a good correlation between expression of p53 protein and histologic grade of lesions. Increased expression of p53 in dysplastic and malignant lesions compared to non dysplastic lesions suggests that p53 protein accumulation may be an early event in carcinogenesis.

The basement membrane and tumor progression in the uterine cervix.

Immunocytochemical localization of the basement membrane (BM) proteins laminin, type-IV collagen and fibronectin were analyzed in normal cervical epithelium, low grade squamous intraepithelial lesions (SILs), high grade SILs and invasive squamous cell carcinoma (SCC) of the uterine cervix. A regular, thick and continuous BM was present in normal cervical epithelium and low grade SIL. Interruptions and discontinuity of the BM were more evident in high grade SILs. There was a good correlation between increasing severity of the lesion and increasing number of breaks. In SCC, the distribution of laminin, collagen IV and fibronectin was related to the degree of cellular differentiation, with decreased immunoreactivity being evident in moderately and poorly differentiated tumors. As the invasive potential of the tumor increased, the fragmentation and loss of BM was more evident. Fibronectin showed only moderate to mild immunoreactivity in normal cervical epithelium and low grade SILs. However, the intensity of expression increased in high grade SILs especially in the peritumoral stroma. It may therefore be concluded from these results that snythesis and reabsorption of BM proteins may be related to shifts in cellular metabolism during tumorigenesis.

Expression of epidermal growth factor in gestational trophoblastic disease (GTD).

Gestational Trophoblastic Disease (GTD) is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of the disease is vague and methods to determine the malignant potentialis limited. Placentae of normal (50) and molar pregnancy (95) including 32 patients showing persistent disease were used in this study. EGF expression was analyzed by immunohistochemistry using monoclonal antibody to EGF and quantitated using isotope labelled antibody to EGF. EGF was expressed more strongly in molar placentae in all gestational ages in comparison with normal placentae of similar gestational age, the expression being restricted mainly to first and second trimesters in normal placentae. Moles with early presenting symptoms (< 12 weeks) were at a higher risk for developing persistent disease. In conclusion, the immunohistochemical study shows high expression of EGF in early normal placentae and moles linking its role to proliferative and differentiating activity of trophoblasts. Tumors with histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGF and thus EGF has the potential of identifying high risk lesions.

Increased expression of cytokeratins 14, 18 and 19 correlates with tumor progression in the uterine cervix.

The expression of cytokeratins (CKs) in normal cervical epithelium, low grade squamous intraepithelial lesions (SIL), high grade SILs and squamous cell carcinoma (SCC) were analyzed using four different monoclonal antikeratin antibodies. In normal cervical epithelium, CK 18 showed strong immunoreactivity in basal and parabasal layers. CK 19 and 14 were expressed only in the basal layer while CK 13 was found selectively n the spinal cells. As the lesions progressed from low grade SIL to high grade SIL, immunoreactivity of CK 18, 19 and 14 in the basal cell compartment increased while the expression of CK 13 decreased. In SCC, as well-differentiated tumors showed decreased immunoreactivity for CK 18, 19 and 14 with CK 13 showing a strong and focal (localized) immunoreactivity. Undifferentiated carcinomas totally lacked CK 13 reactivity. Our findings therefore suggest that expression of CK 18, 19 and 14 may be directly related to tumor grade and CK 13 may be a marker of differentiation in cervical lesions.

c-erbB-2 oncoprotein and epidermal growth factor receptor in cervical lesions.

Epidermal growth factor receptor (EGF-R) and the oncogene product of c-erbB-2 have been shown to be expressed in human tumors and in some cases relate to clinical outcome. This study investigates the correlation between the presence of these receptor proteins and various histological grades of cervical tissues. Immunocytochemical analysis of benign, premalignant and malignant cervical tissues showed a highly significant correlation between the expression of the two proteins and the histological grade of the lesions. Moreover, the overall frequency of coexpression of these proteins was similar. This study suggests that these proteins may be involved in cellular proliferation and have a significant role in the progression of cervical cancer.

Involucrin and tumor progression in the uterine cervix.

The expression of involucrin, a cytoplasmic protein synthesized during squamous maturation, was assessed by immunocytochemistry in different grades of cervical lesions. In normal/benign cervical epithelium and low-grade squamous intraepithelial lesions [SILS or cervical intraepithelial neoplasia (CIN)-1] involucrin showed intense and homogenous cytoplasmic expression in the spinal layers of 75 and 57% of samples, respectively. The basal cell layers showed no expression of involucrin. In high-grade SILs (CIN-2/3) 40% of the samples showed diffuse and focal cytoplasmic expression of involucrin in the differentiated basaloid cells. In the squamous cell carcinomas (SCCs) analyzed, well-differentiated tumors showed intense focal expression in 61% of the cases, moderately differentiated SCCs showed intense expression in 33% of the cases, while poorly differentiated SCCs (PDSCC) showed only a mild focal expression in 7% of cases. With increasing severity of the lesions, patchy expression of involucrin with a mixture of reactive and nonreactive cells predominated. Patterns of immunocytochemical staining for involucrin in cervical lesions of different grades, from low-grade to high-grade SILs, and invasive carcinoma may be of critical importance, if loss of involucrin expression is used as a criterion for neoplastic transformation in cervical epithelium. Our findings suggest that involucrin may be a sensitive marker in identifying the differentiation status of the lesion while the absence of involucrin in PDSCC may be helpful in differential diagnosis.

Tissue binding pattern of plant lectins in benign and malignant lesions of thyroid.

N-acetyl D-galactosamine specific lectins were isolated from the seeds of Jack Fruit (Artocarpus integrifolia) and Winged bean (Psophocarpus tetragonolobus) and D-galactose specific lectin was isolated from peanut (Arachis hypogaea). These lectins were conjugated to Horse Radish Peroxidase (HRP) and were used to study the lectin binding properties of benign and malignant lesions of the thyroid. For comparison of the results 10 normal fresh autopsy specimens were included in the study. The Peanut lectin (PNL) and Jack fruit lectin (JFL) conjugates showed positive binding with the cells in different lesions, while Winged Bean Lectin (WBL), despite its having a common inhibitory sugar, showed no binding even after neuraminidase treatment. These lectins revealed difference in the composition of glycoconjugates of benign and malignant thyroid cells. The HRP conjugated JFL and PNL may be of use in distinguishing carcinomatous tissues from benign tissues which makes them potential tools in the differential diagnosis of thyroid lesions.

Nitrogenase synthesis in Klebsiella pneumoniae: enhanced nif expression without accumulation of guanosine 5'-diphosphate 3'-diphosphate.

Derepression of nitrogen fixation (nif) genes in Klebsiella pneumoniae following transfer from NH+4-sufficiency to N-free medium was preceded by rapid expansion of the guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool. When derepressed in N-free medium supplemented with glutamine (600 micrograms ml-1), expression from the nifH and nifL promoters, determined as beta-galactosidase activity in nif::lac merodiploid strains, was stimulated 7-fold and nitrogenase activity 26-fold; ppGpp did not accumulate, remaining at the levels found in NH+4-repressed populations. The relaxed mutant K. pneumoniae relA40, which accumulates only very low levels of ppGpp, showed partial derepression of nitrogenase activity in the presence of glutamine, thus ppGpp is unlikely to be an effector of nif expression. ATP and GTP levels were elevated under conditions where nif expression was enhanced, consistent with previous data suggesting that maintenance of ATP levels is a prerequisite for the expression of nif genes in K. pneumoniae.