[Clinical analysis of dual enhanced antiplatelet therapy after cerebrovascular intervention for reducing the risk of cerebral infarction recurrence].

To investigate the clinical assessment of dual-enhanced antiplatelet therapy after cerebrovascular intervention to reduce the risk of cerebral infarction recurrence, and to provide a reference for the prevention and treatment of cerebral infarction recurrence risk. 202 patients with cerebral infarction who underwent cerebrovascular intervention in Tianjin Fifth Central Hospital from January 2018 to October 2022 were selected as study subjects. The patients were divided into a treatment group (n=104) based on randomized controlled single-blind method with 61 males and 43 females with a mean age of (62.33±2.57) years old and a control group (n=98) with 56 males and 42 females with a mean age of (62.49±2.36) years old. The control group was given aspirin mono-antiplatelet therapy, and the treatment group was given clopidogrel doublet augmented antiplatelet therapy on the basis of the control group, and both groups continued the treatment for 2 months. Platelet counts, coagulation indexes and inflammatory factors were compared between the two groups before and after treatment, and the America National Institutes of Health Stroke Scale (NIHSS) score was used to assess the neurological functions of the two groups before and after treatment, and the recurrence of cerebral infarction in the two groups was counted within 6 months after treatment. In addition, the patients in the treatment group were divided into the cerebral infarction recurrence group and the cerebral infarction non-recurrence group according to whether they had cerebral infarction recurrence within 6 months after treatment, and the clinical data of the patients in the treatment group were collected to analyze the influencing factors of the dual-enhancement antiplatelet therapy for the recurrence of cerebral infarction in patients with cerebral infarction after cerebral vascular intervention by multifactorial logistic regression. The results showed that after treatment, patients in the treatment group had an international normalized ratio (INR) of (1.76±0.38), a platelet activation rate of (39.52±4.79)%, a platelet aggregation rate of (48.54±5.21)%, a tumor necrosis factor-alpha (TNF-alpha) of (28.37±4.47)ng/L, an interleukin 6 (IL-6) of (24.77±3.52)ng/L, a high-sensitivity C-reactive protein (hs-CRP) of (7.39±1.53)mg/L and an NIHSS score of (6.11±1.39) were lower than those of the control group (2.32±0.41), (44.81±6.37)%, (51.39±5.58)%, (39.66±4.51) ng/L, (29.25±4.04) ng/L, (9.03±1.78) mg/L and (9.93±1.46) points (all P<0.05). At 6-month follow-up of all patients, cerebral infarction recurred in 16 (15.38%) patients in the treatment group and in 33 (33.67%) patients in the control group (χ2=9.185, P<0.05). Kaplan-Meier results showed a statistically significant difference in the rate of recurrence without cerebral infarction in the treatment group compared with the control group(LogRank χ2=4.595,P<0.05). Logistic regression analysis showed that smoking history, cervical vascular plaque, post-treatment NIHSS score, post-treatment stenosis score, post-treatment INR, post-treatment hs-CRP and CYP2C19 gene polymorphism were independent influences on the recurrence of cerebral infarction in cerebral infarction patients with cerebral vascular interventions followed by doublet augmentation of antiplatelet therapy (all P<0.05). In conclusion, dual-enhanced antiplatelet therapy may be an effective measure to reduce the risk of cerebral infarction recurrence after cerebrovascular intervention in patients with cerebral infarction, but it is still influenced by more factors.

[Left occipital dural arteriovenous fistulas presenting as right red eye: a case report].

A 63-year-old male with a healthy history presented with a red and swollen right eye for 3 months. Neuro-ophthalmic examination showed slight bulging of the right eyeball, and multiple spiral conjunctival vessels were visible on the surface of the right conjunctiva, suggesting a right carotid cavernous fistula. Cerebral angiography showed left occipital dural arteriovenous fistulas. After endovascular embolization treatment, the patient's abnormal craniocerebral venous drainage and right eye syndrome resolved, and there was no recurrence during the one-month clinical follow-up after surgery.

Effect analysis of sacral canal therapy combined with Fufang Wulingzhi Tangjiang in the treatment of residual root pain after lumbar surgery.

OBJECTIVE: To observe the clinical effect of a combination of traditional Chinese and western medicine (sacral canal therapy combined with compound Fufang Wulingzhi Tangjiang) in the treatment of residual root pain after lumbar surgery. PATIENTS AND METHODS: From January 2019 to December 2020, 538 patients with residual root pain due to lumbar degenerative diseases were treated in our hospital [open decompression discectomy (ODD), Percutaneous Endoscopic Lumbar Discectomy (PELD) or Transforminal Lumbar Interbody Fusion (TLIF)]. They were randomly divided into control group (basic treatment + celecoxib), observation group 1 (basic treatment + compound Fufang Wulingzhi Tangjiang), observation group 2 (basic treatment + sacral canal therapy) and observation group 3 (basic treatment + sacral canal therapy + Fufang Wulingzhi Tangjiang). Follow-up 3-12 months. The therapeutic effect, VAS score, JOA score, treatment cost, complications, serum interleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-α) were recorded and compared before treatment, 1 week after treatment, 2 weeks after treatment, 1 month after treatment, and the last follow-up. RESULTS: The treatment effect, VAS score, JOA, and treatment cost in the observation group were better than those in the control group (p < 0.05). There were significant differences in the above-mentioned indexes between the observation group 3 and the control group, observation group 1, and observation group 2 (p < 0 01). Inflammatory factors (IL-6, IL1, TNF-α) in the observation group were lower than those in the control group (p < 0 05). Inflammatory factors in observation group 3 were significantly lower than those in the control group, observation group 1, and observation group 2 (p < 0 01). CONCLUSIONS: Sacral canal injection combined with Fufang Wulingzhi Tangjiang can be effective in the treatment of postoperative root pain of lumbar degenerative diseases, which can reduce inflammatory factors such as IL-6, IL-1ß and TNF-α. It has the advantages of quick effect, short treatment time, low cost, high safety, in line with the concept of ERAS, easily accepted by patients and their families, and worthy of further popularizing and applying in clinic.

Decreased long noncoding RNA ADIPOQ promoted cell proliferation and metastasis via miR-219c-3p/TP53 pathway in colorectal carcinoma.

OBJECTIVE: To investigate the expression of Long non-coding RNA ADIPOQ and its facilitating effects on proliferation and invasion of colorectal cancer by modulating the expression of TP53 via sponging with miR-219c-3p. PATIENTS AND METHODS: qRT-PCR was performed to detect the expressions of ADIPOQ and TP53 in human colorectal cancer tissues and cells. CCK-8 assay was performed to evaluate the Caco-2 cells proliferation and transwell assay was performed to evaluate the Caco-2 cells migration. The relationship between ADIPOQ and miR-219c-3p was detected by statistical analysis. Target prediction and Luciferase activity assay were conducted to investigate the binding site and interaction between ADIPOQ and miR-219c-3p. Further, we cloned the mice TP53 3'-UTR into the Luciferase reporter vector and constructed miR-219c-3p binding mutants to verify the inhibited regulation of miR-219c-3p to the TP53 expression. RESULTS: The results suggested that the expression of ADIPOQ and TP53 was downregulated in human colorectal cancer tissues and Caco-2 cells. qRT-PCR and CCK-8 assay showed that ADIPOQ expression is correlated with the proliferation of colorectal cancer cells. Transwell assay showed that ADIPOQ regulated the migration ability of colorectal cancer cells. The bioinformatics prediction and Luciferase assay demonstrated that ADIPOQ serves as ceRNA for miR-219c-3p to further regulate the expression of TP53. CONCLUSIONS: For the first time, we found that lncRNA-ADIPOQ was downregulated in human colorectal cancer cells, which could facilitate tumor proliferation, migration and invasion as a ceRNA by sponging with miR-219c-3p.

Association of genetic variations in miR-146a rs2910164 and miR-149 rs11614913 with the development of classic Kaposi sarcoma.

Classic Kaposi sarcoma is a type of vascular proliferative inflammatory disease. Previous studies have reported significant associations between microRNAs expression and the development of classic Kaposi sarcoma. Here, we conducted a case-control study to investigate the association between miR-146a and miR-149 genetic polymorphisms and risk of classic Kaposi sarcoma in a Chinese population. Both classic Kaposi sarcoma patients and healthy controls were recruited between December 2013 and October 2015. Genotyping of miR-146a and miR-149 was performed by polymerase chain reaction-coupled with restriction fragment length polymorphism. Results showed that the GG genotype of miR-146a was associated with increased risk to classic Kaposi sarcoma (OR = 6.00, 95%CI = 1.19-30.12), as compared with the CC genotype. In the recessive model, we found that the GG genotype carried a 4.55-fold increased risk to classic Kaposi sarcoma as compared with the CC + CG genotype (OR = 2.06, 95%CI = 1.04-20.29). In conclusion, our study demonstrated that miR-146a, but not miR-149 polymorphism, is associated with risk to classic Kaposi sarcoma in the Chinese population.

Microwave irradiation enhances gene and oligonucleotide delivery and induces effective exon skipping in myoblasts.

Microwave (MW) energy consists of electric and magnetic fields and is able to penetrate deep into biological materials. We investigated the effect of MW (2450 MHz) irradiation on gene delivery in cultured mouse myoblasts and observed enhanced transgene expression. This effect is, however, highly variable and critically dependent on the power levels, duration and cycle conditions of MW exposure. MW irradiation greatly enhances delivery of 2'O methyl-phosphorothioate antisense oligonucleotide (AON) targeting mouse dystrophin exon 23 and induces specific exon skipping in cultured myoblasts. Effective delivery of AON by MW irradiation is able to correct the dystrophin reading frame disrupted by a nonsense point mutation in the H2K mdx myoblasts, resulting in the restoration of dystrophin expression. MW-mediated nucleic acid delivery does not directly link to the increase in system temperature. The high variability in gene and oligonucleotide delivery is most likely the result of considerable irregularity in the distribution of the energy and magnetic field produced by MW with the current device. Therefore, achieving effective delivery of the therapeutic molecules would require new designs of MW devices capable of providing controllable and evenly distributed energy for homogenous exposure of the target cells.

Non-viral gene delivery in skeletal muscle: a protein factory.

Ever since the publication of the first reports in 1990 using skeletal muscle as a direct target for expressing foreign transgenes, an avalanche of papers has identified a variety of proteins that can be synthesized and correctly processed by skeletal muscle. The impetus to the development of such applications is not only amelioration of muscle diseases, but also a range of therapeutic applications, from immunization to delivery of therapeutic proteins, such as clotting factors and hormones. Although the most efficient way of introducing transgenes into muscle fibres has been by a variety of recombinant viral vectors, there are potential benefits in the use of non-viral vectors. In this review we assess the recent advances in construction and delivery of naked plasmid DNA to skeletal muscle and highlight the options available for further improvements to raise efficiency to therapeutic levels.

Expression of two related viral early genes in Epstein-Barr virus-associated tumors.

The transcription of two early "leftwardly" expressed genes carrying repetitive sequences, IR2 and IR4, has been studied for Epstein-Barr virus-associated tumors, and for established B-cell lines, using sequence-specific probes generated for this purpose. Whereas the IR4 transcript was identified in every tumor and cell line assessed (except B95-8, with a deletion that removes the gene), expression of the IR2 gene was restricted to B lymphocytes. Though the promoters for both transcripts lie within homologous regions (D(L) and D(R)) in the viral genome, the IR2 promoter appears more tightly regulated. Detailed characterization of the IR4 transcript from a nasopharyngeal carcinoma tumor, C15, identifies a sequence variant of this gene that differs from those reported for B cells; in situ hybridization methods show transcription to be restricted to a subset of cells, with the strongest signals seen adjacent to host stroma. As with B cells in culture (Y. Gao, P. R. Smith, L. Karran, Q. L. Lu, and B. E. Griffin, J. Virol. 71:84-94, 1997), chemical induction enhanced transcriptional expression of the IR4 gene in the C15 tumor, although staining for both the IR4 antigen and that of the virus lytic switch, Zta, gave negative results. In a Burkitt's lymphoma biopsy specimen, however, both proteins were found expressed, notably in the same subset of cells. The data here and elsewhere (Gao et al., J. Virol., 1997) are consistent with a block to intracellular transport of the transcript(s) and suggest nuclear roles for it in tumors, possibly in RNA processing and viral lytic replication. Both roles could be fulfilled in the absence of translation.

Decreased HLA-A expression in prostate cancer is associated with normal allele dosage in the majority of cases.

A comparison has been made of the phenotypic expression of MHC class I antigens with the corresponding HLA-A genotype in 15 cases of benign prostatic hyperplasia (BPH) and 34 cases of primary locally invasive prostatic carcinoma. Expression of class 1 protein, detected by immunocytochemistry, was partially or completely lost in approximately 90% of the tumours examined. Comparative genomic analysis of the beta2 microglobulin (beta2m) gene and 15 individual HLA-A haplotypes using a polymerase chain reaction (PCR)-based method demonstrated abnormal gene dosage in the minority of cases: homozygous deletion of the beta2m locus was detected in one case and HLA-A allele in two cases (HLA-A1 and HLA-A2, respectively), representing approximately 8% of the population studied. This first comparative study of gene dosage and expression of class 1 protein reported for prostate cancer reveals that deletion is not the cause of the partial or complete loss seen in the majority of cases. This raises the possibility, in the future, for novel selective immunomodulatory therapeutic strategies which stimulate a clinically significant re-expression of class 1 protein and associated cytotoxic T-cell response.

A new blocking method for application of murine monoclonal antibody to mouse tissue sections.

Antigen detection with primary antibody of the same species as the test tissue is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. This severely limits the use of murine monoclonal antibodies on tissues of the mouse, the most widely used experimental model system; no method for blocking this is fully satisfactory. Here we show that background staining encountered in this system results largely from the binding of secondary antibodies via both Fc and Fab to endogenous immunoglobulins and other tissue components. A simple and efficient blocking strategy was established, employing papain-digested whole fragments of unlabeled secondary anti-mouse Igs enriched with Fc fragment of the same Igs. We have used this method to visualize dystrophin, an antigen expressed at low level, in revertant fibers of mdx mouse by both immunoperoxidase and immunofluorescence methods. In combination with the use of a biotin-streptavidin immunohistochemical detection protocol with biotinylated anti-mouse F(ab')2 as second layer, we eliminated the heavy background in this system and achieved strong signal amplification to demonstrate the specific antigen clearly. Double labeling with one mouse antibody and one antibody from another species was performed without signal interference. This principle can be adapted for wider applications, such as antibodies of other species on homologous tissues and perhaps where high background is found with heterologous antibodies. (J Histochem Cytochem 46:977-983, 1998)

Expression of bcl-2 in bladder neoplasms is a cell lineage associated and p53-independent event.

AIMS: To investigate bcl-2 and p53 protein expression in hyperplastic, metaplastic and neoplastic epithelia of the urinary bladder in relation to cell lineages (transitional versus glandular epithelia). METHODS: Formalin fixed, paraffin wax embedded archival tissue blocks of 29 transitional cell carcinomas (TCC), 11 adenocarcinomas, five specimens of cystitis glandularis, four papillomas, and seven samples of morphologically normal bladder mucosa were examined immunohistochemically with antibodies specific to bcl-2 and p53. Consecutive sections were used to assess co-expression of the two proteins. RESULTS: bcl-2 protein was expressed heterogeneously in basal cells of the normal transitional epithelium, whereas p53 was rarely detectable in either normal or hyperplastic epithelium. Of the 29 TCCs, 20 (69%) expressed immunodetectable p53 which was positively associated with grade. In contrast, bcl-2 was detected in four (14%) TCCs and its expression was not associated with grade. bcl-2 was expressed constitutively in all five specimens of cystitis glandularis and in all adenocarcinomas; p53 was co-expressed in most of the latter. There was no association between bcl-2 and p53 protein expression in the TCCs. Expression of bcl-2 protein correlated negatively with grade of adenocarcinoma. CONCLUSION: In bladder adenocarcinomas, bcl-2 expression correlated negatively with tumour grade whereas p53 was associated positively with tumour grade. The association of bcl-2 with cystitis glandularis and adenocarcinoma but not TCC suggests that it may be involved in triggering a lineage switch converting transitional epithelium to a glandular phenotype.

Expression of bcl-2 and p53 oncoproteins in schistosomiasis-associated transitional and squamous cell carcinoma of urinary bladder.

OBJECTIVES: To analyze the expression of bcl-2 and p53 proteins in schistosomiasis-associated bladder cancer compared to adjacent urothelium showing morphological alteration including hyperplasia, metaplasia and dysplasia. MATERIALS AND METHODS: Twenty-two formalin-fixed and paraffin-embedded samples of histologically confirmed schistosomiasis-associated bladder tumours were assessed for grade, pathological stage (pT category) and the presence of hyperplasia, metaplasia and dysplasia in adjacent mucosa. There were 11 transitional cell carcinomas (TCCs), 10 squamous cell carcinomas (SCCs) and one diffuse infiltrating (signet-ring cell type) adenocarcinoma. Epithelial hyperplasia was observed in seven cases and metaplasia in 18, most of which were squamous except for a single case of glandular metaplasia. Focal dysplastic areas were observed in eight cases. Sections were stained immunohistochemically for the expression of bcl-2 and p53 proteins. RESULTS: Immunoreactivity for bcl-2 was present in seven of 22 cases (four SCCs and three TCCs) of which three cases (two SCCs and one TCC) were strongly positive, but in contrast to previous studies, there was no increase in the poorly differentiated tumours, bcl-2 was absent in metaplastic and dysplastic epithelium (except in a single case of glandular metaplasia) but it was present at low levels in basal cells of morphologically normal or hyperplastic transitional epithelium in 15 of 22 cases. Nine of 11 TCCs and seven of 10 SCCs showed p53 nuclear immunoreactivity. Heterogenous weak to moderate immunoreactivity for p53 was seen in 13 of 18 cases of metaplasia and in seven of eight cases of dysplasia in the mucosa adjacent to tumours, p53 was absent in normal and hyperplastic urothelium. Co-expression of p53 and bcl-2 was only seen in well differentiated areas of three tumours (two SCCs and one TCC). CONCLUSION: This study detected the expression of bcl-2 in a subset of bladder cancers (32%), whereas most (72%) of the tumours expressed immunoreactive p53. Additionally, p53 was also detected in metaplastic and dysplastic epithelium. Over-expression of both p53 and bcl-2 in the same tumour was only evident in a minority of schistosomiasis-associated cancers (13%). There was no inverse relationship of p53 and bcl-2 immunoreactivity.

Human papillomavirus 16 and 18 infection is absent in urinary bladder carcinomas.

OBJECTIVE: The role of human papillomavirus (HPV) in the pathogenesis of bladder neoplasia remains controversial. Studies to date have mainly utilized polymerase chain reaction and DNA blot techniques to detect HPV DNA. The detection rates have varied from 0 to 81%. One of the major limitations of these techniques is that they lack topographic information. We have used a highly sensitive in situ hybridization (ISH) technique which detects single copies of viral genome and provides topographic information for investigation of HPV infection in bladder carcinomas. MATERIALS AND METHODS: Thirty-one samples of formalin-fixed paraffin-embedded bladder carcinomas (4 adenocarcinomas, 5 squamous cell and 22 transitional cell carcinomas) were examined using non-isotopic ISH with biotin-labelled DNA probes of HPV 16 and 18 subtypes. HPV-positive skin and anal carcinoma samples were used as controls. RESULTS AND CONCLUSIONS: No positive signals for HPV 16 or 18 were detected in any of the bladder carcinoma samples. Given the sensitivity of the technique, the result indicates that HPV 16 and 18 are not implicated in the development of bladder neoplasia compared to certain other epithelial malignancies. The possibility that other subtypes of HPV contribute to bladder epithelial carcinogenesis remains to be clarified.

Induction of an exceptionally high-level, nontranslated, Epstein-Barr virus-encoded polyadenylated transcript in the Burkitt's lymphoma line Daudi.

An Epstein-Barr virus transcript (designated D-HIT [Daudi high-level-inducible transcript]), constitutively expressed at low levels in the Burkitt's lymphoma (BL)-derived cell line Daudi, can be induced with tetradecanoylphorbol acetate or n-butyrate or, in combination, to about 1% of the levels of high-molecular-weight RNAs in cells. The transcript can also be induced in some other EBV-positive BL-derived cells but to a much lesser extent, particularly in lines that can give rise to productive infection. D-HIT is viral in origin and is composed largely of repetitive sequence. It is polyadenylated but mainly nuclear in location and is highly structured, sensitive only to double-strand-specific RNase. It is endogenously expressed in interferon-sensitive Daudi strains but not in an insensitive strain, Daudi 100K. D-HIT contains a part of a viral open reading frame (designated LF3, and deleted in the prototype B95-8 strain), using an internal polyadenylation (AAUAAA) sequence as a signal to specify processing of its 3' end. In Daudi cells, the promoter contains a putative hinge structure, as found in some interferon-inducible genes and c-myc. Since D-HIT lies adjacent to, probably even encompassing, one of the two viral lytic origins (D(R)) of replication, it may have a role in the regulation of DNA replication. Alternatively, or in addition via its double-stranded structure, D-HIT may play a regulatory role in interferon pathways. Its promoter could be of value for studying expression in constructions containing heterologous genes.

Properties of HPV-positive and HPV-negative anal carcinomas.

Evidence of human papillomavirus (HPV) can be found in up to 85 per cent of anal carcinomas. In the vulva, a discrete subset of HPV-positive carcinomas which show koilocytic morphology and distinct clinical features has recently been identified (warty carcinoma). The morphological and prognostic features of HPV-positive and HPV-negative anal carcinomas were compared in this study of the tumour distribution of HPV DNA. Vulval and anal neoplasia are similar in many ways and we have also looked to see if their similarity extends to 'warty' morphology in relation to HPV status. Thirty-five resection specimens of anal carcinoma were examined with biotin-labelled probes for HPV 6, 11, 16, and 18 DNA, using a non-isotopic in situ hybridization (ISH) technique. No tumour was found to contain HPV 6, 11, or 18. Twenty-four (72 per cent) showed positivity for HPV 16 DNA. Staining was homogeneous and independent of local squamous, basaloid, or ductal differentiation. The majority of tumours showed staining suggestive of episomal, non-productive HPV infection. HPV-positive tumours were more likely to occur in the anal canal than perianally and to show a mixed squamous and basaloid appearance. No difference between the two groups was found in patient age, presence of adjacent dysplasia, ductal differentiation, or prognosis. There was no correlation between condylomatous tumour morphology and HPV 16 DNA positivity; thus, a subset equivalent to vulval warty carcinoma could not be identified.

bcl-2: role in epithelial differentiation and oncogenesis.

The precise regulation and maintenance of balance between cell proliferation and cell death in multicellular organisms is critical for tissue homeostasis. bcl-2 initiates a new gene family involved in the regulation of cell death and survival without affecting cell proliferation. Expression of Bcl-2 has been reported in a wide range of hematopoietic cells, nonneoplastic epithelia (both hormone-responsive and nonresponsive), and epithelial malignancies. Although the major group of epithelial cells expressing Bcl-2 protein are in the proliferating zones, expression is not directly related to cell proliferation. Bcl-2 is also associated with stem cells committed to differentiation and morphogenesis. The survival advantage provided by Bcl-2 prolongs the life span of epithelial cells with differentiation potential and allows proliferation, differentiation, and morphogenesis to proceed. The gene expression in hormone-responsive organs may contribute to the sustained life of those terminally differentiated epithelial cells and a decrease in Bcl-2 levels leads to cell death by apoptosis. Overexpression of bcl-2 protects epithelial cells from death, but it is neither able to immortalize normal cells, nor to cause tumorigenic transformation of immortalized epithelial cells. Heterogeneous expression of Bcl-2 in epithelial malignancies suggests that the gene is differentially regulated. Furthermore, its expression in association with precancerous lesions suggests a role in the early stage of tumorigenesis. The effects of Bcl-2 expression on sensitivity of epithelial cells to drug, radiation, and hormone therapies vary depending on the typed of tumor. Expression of Bcl-2 is associated with resistance to hormone therapy and recurrence in prostate carcinomas, whereas in lung and breast carcinomas it is associated with a better prognosis. Studies now being performed should clarify the underlying mechanisms of differential gene regulation in different tissues and show the clinical significance of the expression of bcl-2 and other members of the bcl-2 gene family.

Nuclear and cytoplasmic bcl-2 expression in endometrial hyperplasia and adenocarcinoma.

The bcl-2 proto-oncogene, which inhibits programmed cell death (apoptosis), has recently been found to be cyclically expressed in human endometrium. In order to investigate its role in endometrial hyperplasia and neoplasia, bcl-2 expression was studied in 25 cases of endometrial carcinoma and 20 cases of endometrial hyperplasia (eight simple, two complex, and ten atypical hyperplasias). Uniform intense cytoplasmic bcl-2 expression was found in all cases of non-atypical hyperplasia, and less strong positivity in eight out of ten cases of atypical hyperplasia. In well-differentiated carcinomas, nine out of ten showed weak to moderate bcl-2 expression, whereas six out of seven poorly differentiated carcinomas were bcl-2-negative. Moderately differentiated tumours were an intermediate group, with six out of eight being positive. Widespread localization of bcl-2 protein to the chromosomes of dividing cells was also demonstrated, regardless of cytoplasmic bcl-2 expression, with rare staining of interphase nuclei. Our findings suggest a role for bcl-2 in the natural history of endometrial neoplasia and studies are needed to determine its usefulness as a prognostic marker. The finding of bcl-2 localization to chromosomes has important implications for its mode and site of action.

Survival of allogeneic cells in cultured organotypic skin grafts.

Organotypic cultures of human skin were made using dermal fibroblasts seeded into a type I collagen gel overlaid with epidermal keratinocytes. Full-thickness excision of tattoos was performed on five patients, three of whom received sex-mismatched allografts. Patients were not immunosuppressed. Biopsies were obtained up to 3.5 years later. In situ hybridization of the PHY2.1 repetitive Y chromosome sequence revealed male fibroblasts and keratinocytes at 11 weeks and 2.5 years in the two female patients grafted with male cells. Structural components in the dermal substitute matured with time, and elastic fibers formed an interlacing meshwork by 18 months. Electron microscopy of the dermal-epidermal junction of an organotypic allograft revealed anchoring fibrils that had normal features at this time. Hyperemia of early grafts settled and contour correction was maintained, while repigmentation was variable. Hypertrophic scars did not occur, and graft contracture was never more than 20 percent. We conclude that this organotypic skin graft shows potential toward the goal of allogeneic skin replacement in a one-step procedure.

bcl-2 overexpression inhibits cell death and promotes the morphogenesis, but not tumorigenesis of human mammary epithelial cells.

Overexpression of the B cell leukemia/lymphoma-2 (bcl-2) gene has been shown to confer a survival advantage on cells by inhibiting apoptosis. In epithelia, the bcl-2 gene is also related to development and differentiation, and the protein is strongly expressed in the embryo in the epithelial cells of the developing mammary gland. To investigate directly the effect of bcl-2 on human epithelial cells, we used an amphotropic recombinant retrovirus to introduce the gene into nontumorigenic cell lines developed from luminal epithelial cells cultured from milk. Here we demonstrate that while bcl-2 overexpression does not directly induce the tumorigenic phenotype, it provides a survival advantage to the mammary epithelial cells by inhibiting cell death at confluence or under conditions of serum starvation, bcl-2 can also affect the phenotype of the original epithelial cells, and promote epithelial-mesenchymal conversion, accompanied by loss of the cell adhesion molecules E-cadherin and alpha 2 beta 1 integrin. The extent of the epithelial-mesenchymal conversion varies with small differences in the phenotype of the parental line and with the level of expression of Bcl-2 and in some cases cell lines emerge with a mixed phenotype. The increased survival of Bcl-2-expressing cells at confluence results in multilayering, and the development of three- dimensional structures. Where a mixed phenotype is observed these structures consist of an outer layer of polarized epithelial cells separated by a basement membrane-like layer from an inner mass of fibroblastoid cells. Branching morphogenesis of bcl-2 transfectants is also observed in collagen gels (in the absence of fibroblast growth factors). The results strongly indicate that by increasing their survival under restrictive growth conditions, and by modifying the epithelial phenotype, bcl-2 can influence the specific morphogenetic behavior of mammary epithelial cells.

Criteria for consistent and high sensitivity of DNA in situ hybridization on paraffin sections: optimal proteolytic enzyme digestion.

It is technically challenging for the detection of target DNA in low abundancy, such as viral DNA sequences in latently infected cells by nonisotopic in situ hybridization (ISH). Consistent result is even more difficult to achieve on routine paraffin sections. Proteolytic enzyme digestion is most critical for both consistency and sensitivity of the technique. We here have investigated the effect of enzyme digestion on cell morphology, protein and DNA reduction, and hybridization efficiency. The results demonstrated that enzyme digestion improves efficiency of ISH through a process involving partial DNA purification on sections. There is a clear relationship between proteolytic enzyme digestion, morphology changes, and hybridization efficiency. Although detection of DNA sequences in abundance can be achieved within a relatively wide range of digestion levels, maximum hybridization efficiency was always related to the cells, which showed morphology of nuclear swollen, weak homogeneous chromatin staining with hematoxylin and loss of visible nuclear membrane. Detection of viral DNA in low copy number critically depends on the creation of the morphologic changes by enzyme digestion. The morphological changes would therefore serve as important criteria for optimal digestion, result interpretation, and comparison.