Biofiltration of airborne VOCs with green wall systems-Microbial and chemical dynamics.

Botanical air filtration is a promising technology for reducing indoor air contaminants, but the underlying mechanisms need better understanding. Here, we made a set of chamber fumigation experiments of up to 16 weeks of duration, to study the filtration efficiencies for seven volatile organic compounds (VOCs; decane, toluene, 2-ethylhexanol, α-pinene, octane, benzene, and xylene) and to monitor microbial dynamics in simulated green wall systems. Biofiltration functioned on sub-ppm VOC levels without concentration-dependence. Airflow through the growth medium was needed for efficient removal of chemically diverse VOCs, and the use of optimized commercial growth medium further improved the efficiency compared with soil and Leca granules. Experimental green wall simulations using these components were immediately effective, indicating that initial VOC removal was largely abiotic. Golden pothos plants had a small additional positive impact on VOC filtration and bacterial diversity in the green wall system. Proteobacteria dominated the microbiota of rhizosphere and irrigation water. Airborne VOCs shaped the microbial communities, enriching potential VOC-utilizing bacteria (especially Nevskiaceae and Patulibacteraceae) in the irrigation water, where much of the VOC degradation capacity of the biofiltration systems resided. These results clearly show the benefits of active air circulation and optimized growth media in modern green wall systems.

Plant metallothioneins--metal chelators with ROS scavenging activity?

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins present in plants, animals, fungi and cyanobacteria. In plants, MTs are suggested to be involved in metal tolerance or homeostasis, as they are able to bind metal ions through the thiol groups of their cysteine residues. Recent reports show that MTs are also involved in the scavenging of reactive oxygen species (ROS). The interplay between these roles is not entirely clear. Plants have many MT isoforms with overlapping expression patterns, and no specific role for any of them has been assigned. This review is focused on recent findings on plant MTs.

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens.

To study the role of metallothioneins (MTs) in Zn accumulation, the expression of TcMT2a, TcMT2b, and TcMT3 was analysed in three accessions and 15 F(3) families of two inter-accession crosses of the Cd/Zn hyperaccumulator Thlaspi caerulescens, with different degrees of Zn accumulation. The highest expression levels were found in the shoots of a superior metal-accumulating calamine accession from St Laurent le Minier, with >10-fold TcMT3 expression compared with another calamine accession and a non-metallicolous accession. Moreover, F(3) sibling lines from the inter-accession crosses that harboured the MT2a or MT3 allele from St Laurent le Minier had higher expression levels. However, there was no co-segregation of TcMT2a or TcMT3 expression and Zn accumulation. To examine the functions of TcMTs in plants, TcMT2a and TcMT3 were ectopically expressed in Arabidopsis. The transformant lines had reduced root length in control medium but not at high metal concentrations, suggesting that the ectopically expressed proteins interfered with the physiological availability of essential metals under limited supply. The Arabidopsis transformant lines did not show increased tolerance to Cd, Cu, or Zn, nor increased Cd or Zn accumulation. Immunohistochemical analysis indicated that in roots, MT2 protein is localized in the epidermis and root hairs of both T. caerulescens and Arabidopsis thaliana. The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarily involved in Zn accumulation as such. However, the elevated expression levels in the metallicolous accessions suggests that they do contribute to the metal-adapted phenotype, possibly through improving Cu homeostasis at high Zn and Cd body burdens. Alternatively, they might function as hypostatic enhancers of Zn or Cd tolerance.

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant Thlaspi caerulescens.

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.

Enhanced copper tolerance in Silene vulgaris (Moench) Garcke populations from copper mines is associated with increased transcript levels of a 2b-type metallothionein gene.

Silene vulgaris (Moench) Garcke has evolved populations with extremely high levels of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in S. vulgaris, we screened a cDNA library derived from a highly copper-tolerant population using Arabidopsis-based MT probes and identified an MT2b-like gene. When expressed in yeast, this gene, SvMT2b, restored cadmium and copper tolerance in different hypersensitive strains. Northern-blot analysis and quantitative reverse transcriptase-PCR showed that plants from the copper-tolerant S. vulgaris populations had significantly higher transcript levels of SvMT2b than plants from the copper-sensitive populations, both in roots and shoots and with and without copper exposure. Southern-blot analysis suggested that the higher expression of the latter allele was caused by gene amplification. Segregating families of crosses between copper-sensitive and copper-tolerant plants exhibited a 1 to 3 segregation for SvMT2b expression. Allele-specific PCR showed that low-expression F(3) plants were homozygous for the allele inherited from the copper-sensitive parent, whereas high-expression plants possessed at least one allele from the tolerant parent. SvMT2b expression did not cosegregate with copper tolerance in crosses between sensitive and tolerant plants. However, a significant cosegregation with copper tolerance did occur in families derived from crosses between moderately tolerant F(3) plants with different SvMT2b genotypes. Thus, overexpression of SvMT2b conferred copper tolerance although only within the genetic background of a copper tolerant plant.

Genetic engineering in the improvement of plants for phytoremediation of metal polluted soils.

Metal concentrations in soils are locally quite high, and are still increasing due to many human activities, leading to elevated risk for health and the environment. Phytoremediation may offer a viable solution to this problem, and the approach is gaining increasing interest. Improvement of plants by genetic engineering, i.e. by modifying characteristics like metal uptake, transport and accumulation as well as metal tolerance, opens up new possibilities for phytoremediation. So far, only a few cases have been reported where one or more of these characteristics have been successfully altered; e.g. mercuric ion reduction causing improved resistance and phytoextraction, and metallothionein causing enhanced cadmium tolerance. These, together with other approaches and potentially promising genes for transformation of target plants are discussed.

Body iron stores are associated with serum insulin and blood glucose concentrations. Population study in 1,013 eastern Finnish men.

OBJECTIVE: To study if there is an association between mildly elevated body iron and glucose homeostasis indexes. RESEARCH DESIGN AND METHODS: A cross-sectional population study was conducted in 1,013 middle-aged men, and an association of serum ferritin with concentrations of serum insulin, blood glucose, and serum fructosamine was tested. RESULTS: The mean concentration of fasting serum insulin was 21.6% higher (95% CI 7.3-37.9%, P < 0.001) in the 5th quintile of serum ferritin compared with the 1st quintile. The elevation in blood glucose was 6.1% (95% CI 2.3-9.9%, P < 0.001) and in serum fructosamine 3.9% (1.5-6.9%, P < 0.01). CONCLUSIONS: Mildly elevated body iron stores are associated with statistically significant elevations in glucose homeostasis indexes.

Human papillomavirus infections in vulvar precancerous lesions and cancer.

To elucidate some of the recently arisen issues related to the bimodal disease pattern of vulvar intraepithelial lesions (VIN) and vulvar cancer, a series of 27 consecutive women with vulvar symptoms was analyzed for human papillomavirus (HPV) involvement by colposcopy, light microscopy and in situ hybridization (ISH) for HPV types 6, 11, 16, 18, 31, 33 and 42. Altogether, HPV DNA was discovered in 13/27 (48.1%) of the lesions by ISH; the rest were HPV DNA negative for the seven HPV types tested. HPV DNA was present in both of two exophytic lesions (HPV 6 in condyloma and HPV 16 in verrucous cancer). Of the flat lesions, 7/13 (53.8%) were HPV DNA positive. HPV 6 was confined to low grade lesions (HPV/non-VIN and VIN 1), whereas HPV 11 was found in a case of VIN 3 as well. Of the invasive carcinomas, three of four were HPV DNA positive (2 HPV 16 and 1 HPV 31). Dystrophic changes were detected in three of four invasive carcinomas and in all three HPV 16-positive lesions. Dystrophic changes were absent in 9 of 14 (64.3%) of HPV DNA-negative lesions. Fifty percent (7/14) of vulvar warty lesions (without concomitant VIN) were found in women younger than 60. Three of four invasive carcinomas occurred in women older than 60. This small series provided additional evidence of HPV involvement in the pathogenesis of VIN lesions, and the findings support the hypothesis of a multifactorial etiology in vulvar carcinogenesis in which HPV, dystrophic changes and chronic inflammatory disease play a synergistic role.

Transitional cell carcinoma of the bladder: failure to demonstrate human papillomavirus deoxyribonucleic acid by in situ hybridization and polymerase chain reaction.

Human papillomaviruses have been implicated in the pathogenesis of a variety of malignancies, particularly those of the anogenital tract. Some recent reports on the presence of human papillomavirus in bladder cancer have raised the possibility that it might be involved in the development of this malignancy as well. To study this concept, a series of 108 transitional cell carcinomas of the bladder were screened for the presence of human papillomavirus deoxyribonucleic acid (DNA) by in situ hybridization with biotin-labeled human papillomavirus cocktail probe and polymerase chain reaction with human papillomavirus L1 consensus primers. Although the positive controls showed strong hybridization signals, no evidence for human papillomavirus DNA was found in any of the bladder carcinomas by in situ hybridization. Similarly, despite the amplification of a 450 bp product in cervical human papillomavirus lesions (used as positive controls), no signals were obtained in any of the bladder tumors studied. beta-globin gene sequences (110 bp), serving as internal controls, were consistently amplified from all tumor samples, suggesting that cellular DNAs from the carcinoma specimens were sufficient for the amplification reaction. These data indicate that human papillomavirus infection is rare in transitional cell carcinoma of the bladder. The significance of these findings is discussed in relation to previous reports on human papillomavirus involvement in bladder carcinomas.

Detection of p53 protein and Ki-67 proliferation antigen in human papillomavirus (HPV)-positive and HPV-negative cervical lesions by immunohistochemical double-staining.

In HPV-associated genital lesions, low or absent expression of p53 has been attributed to the rapid degradation of p53 through its binding with HPV E6 protein. In this study, we examined p53 protein expression with two antibodies (CM1 polyclonal and PAb 1801 monoclonal antibodies), and Ki-67 proliferation antigen (monoclonal antibody) using an immunohistochemical (IHC) double-staining technique in 77 HPV-positive cervical lesions (HPV6, HPV11, HPV16, HPV18, HPV31, and HPV33) and in 15 HPV-negative cases. p53 protein expression was detected in 36/92 (39.1%) of the specimens. Of the p53-positive cases, 80.6% (29/36) were HPV-positive samples, including 10/23 (43.5%) of HPV16- and 3/10 (30%) of HPV18-positive biopsies. In 52.8% of the p53-positive samples, the expression was found in less than 5% of the basal cells which were also positive for Ki-67. Ki-67 proliferation marker was found in 91/92 specimens, most intensely in those infected by HPV16. p53 was more abundant in progressive or persistent lesions, but no differences were found between HPV-positive and HPV-negative samples. The positive IHC double-staining of both p53 and Ki-67 proliferation antigen in the same basal (and parabasal) cells indicates that these two normal cell-cycle proteins are being expressed while the cells are entering from the G1 to the S phase of the cell cycle. Since the latter property is only attributed to the wild-type p53 (but not to mutated p53), the p53 protein detected in HPV lesions by IHC is likely to be the wild-type p53 rather than mutated p53, and the result was also confirmed by using p53 mutant specific antibody PAb 240. Accordingly, the concept of HPV inactivating the wild-type p53 protein should be re-examined, and other mechanisms for HPV-mediated carcinogenesis should be considered.

Expression of transforming growth factor-beta 1 and -beta 2 in human papillomavirus (HPV)-associated lesions of the uterine cervix.

Human papillomaviruses (HPV) are implicated in the multistep process of cervical carcinogenesis. Transforming growth factor beta(TGF-beta) inhibits the proliferation of epithelial cells, and it has also been found to inhibit HPV gene expression in nontumorigenic epithelial cell lines. In the present study, we examined the expression of TGF-beta 1 and TGF-beta 2 protein immunohistochemically (IHC) in a series of 95 HPV-positive and HPV-negative lesions of the uterine cervix, with special emphasis on HPV type, grade of cervical intraepithelial neoplasia (CIN), and the clinical course of the disease. Expression of TGF-beta 1 was found in 56/95 (59%) and that of TGF-beta 2 in 87/95 (92%) of the specimens. Cytoplasmic TGF-beta 2 staining was localized in the epithelial layers higher than that of TGF-beta 1, which showed also some nuclear staining and was located in the basal cells of the epithelium as well. TGF-beta 1 was expressed in 36/68 (53%) of HPV-positive samples and in 16/21 (76%) of HPV-negative samples; TGF-beta 2 expression was detectable in 63/68 (93%) and 18/21 (86%), respectively. TGF-beta 1 was present slightly more frequently in HPV-CIN lesions (23/41, 56%) than in HPV-NCIN (HPV without CIN) specimens (13/27, 48%). TGF-beta 2 expression was detected in 39/41 (95%) of HPV-CIN and in 24/27 (89%) of HPV-NCIN specimens. TGF-beta 2 expression was not related to the clinical course of the disease. TGF-beta 1 expression was most frequent in regressed and persistent lesions (> 60%), compared to 45% in progressed and 33% in the recurred lesions. The results suggest that TGF-beta (especially TGF-beta 2) expression is common in CIN lesions, but the pattern and intensity of TGF-beta expression examined by IHC are not clearly related to the grade of the lesions or their clinical course. Assessment of the biological activity of TGF-beta s and their influence on HPV genes may shed more light on HPV-associated carcinogenesis.

Frequent mutations of p53 gene in oesophageal squamous cell carcinomas with and without human papillomavirus (HPV) involvement suggest the dominant role of environmental carcinogens in oesophageal carcinogenesis.

Epidemiological evidence suggests that alcohol intake, use of tobacco, ingestion of mycotoxins and nitrosamines and nutritional deficiencies are high-risk factors for the development of oesophageal cancer. Similarly, viral infections have been postulated to play a role in some tumours. However, the molecular events underlying the development of oesophageal carcinoma are poorly understood as yet. Loss of p53 tumour-suppressor gene function has been found in different human malignancies, and it can occur in a variety of ways, including gene mutation and interaction with the E6 protein of oncogenic human papillomaviruses (HPVs). Because the oesophageal mucosa is potentially exposed to mutagens and HPVs, we studied DNA samples derived from nine HPV-positive squamous cell carcinomas and 12 HPV-negative tumours. Exons 5-9 of the p53 gene containing phylogenetically conserved domains were examined using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique. HPV detection was done using DNA in situ hybridisation with biotin-labelled HPV DNA probes. Mutations were detected in eight (38%) out of the 21 cases. Three mutations were found in exons 5/6, three in exon 7 and two in exon 8/9. Six (50%) of the 12 HPV-negative carcinomas showed p53 mutations. Two (22.2%) of the nine HPV-positive carcinomas were found to contain p53 mutations as well; one contained HPV 16 DNA sequences and showed p53 mutation in exon 8/9, and the other was HPV 6/11 positive with the mutation in exon 5/6. Although mutations were more common in HPV-negative tumours (50.0% vs 22.2%), the difference in p53 mutations in HPV-positive and -negative tumours did not reach statistical significance (P = 0.1946). These data indicate that inactivation of the p53 gene is a frequent event in oesophageal squamous cell carcinomas and such an inactivation might be an important molecular pathway for the development of oesophageal cancer. The findings of p53 mutations in HPV-positive oesophageal carcinomas suggest that HPV and p53 mutation were not mutually exclusive events. The presence of frequent mutations of p53 gene in both HPV-positive and -negative oesophageal carcinomas suggests a dominant role of environmental carcinogens in oesophageal carcinogenesis.

Epidermal growth factor receptor, c-erbB-2 proto-oncogene and estrogen receptor expression in human papillomavirus lesions of the uterine cervix.

The expression of epidermal growth factor receptor (EGF-R), c-erbB-2 proto-oncogene, and estrogen receptor (ER) was studied immunohistochemically in a series of 97 human papillomavirus (HPV) lesions of the uterine cervix, with special emphasis on their association with the HPV type, grade of intraepithelial neoplasia (CIN), and the natural history of the disease. EGF-R expression was found in 95 of 97 (98%) specimens, mainly in basal and parabasal cells. Diffuse nuclear and cytoplasmic staining was detected in 36 of 97 (37%) samples, of which 29 were HPV positive. This staining pattern was most prominent in HPV 18-positive and in CIN lesions. Weak or moderate c-erbB-2 expression was found in 26 of 97 (27%) specimens. Estrogen receptor expression was observed in 28 of 77 (36%) samples, epithelial staining was seen in 11 of 77 (14%), and stromal staining occurred in 24 of 77 (31%) specimens. No clear-cut associations were established between the EGF-R, c-erbB-2, or ER expression and HPV type, nor in CIN or the clinical course of HPV infections. This failure for EGF-R, c-erbB-2, and ER to be associated with the specific HPV types, grade of CIN, or the clinical course of cervical HPV lesions suggests that the assessment of these factors is of limited value in explaining the development of HPV-associated CIN and in predicting the prognosis of this disease.

The state of the p53 gene in human papillomavirus (HPV)-positive and HPV-negative genital precancer lesions and carcinomas as determined by single-strand conformation polymorphism analysis and sequencing.

Human papillomavirus (HPV) is frequently associated with cervical carcinoma. Inactivation of the p53 tumor suppressor gene product by binding to the HPV encoded E6 protein is considered as an important pathway for malignant progress in HPV-infected cells. In contrast, mutations of the p53 gene have been found in HPV-negative cervical carcinoma cells. To evaluate the involvement of p53 inactivation for the development of genital carcinoma, we determined the state of the p53 gene in 20 genital precancer lesions and carcinomas, which had been previously studied for the expression of p53 protein and the presence of HPV DNA. Exons 5 through 9 of the p53 gene were analyzed by single-strand conformation polymorphism analysis of polymerase chain reaction (PCR)-amplified DNA fragments, and the results obtained by the PCR-SSCP analysis were confirmed by DNA sequencing. No mutations were detected in any of the specimens, including the three HPV-negative cases. The present results suggest that the functional inactivation of p53 is not invariably required for the induction of malignant transformation in the genital tract, and thus other genetic events can also significantly participate in genital carcinogenesis.

Tumourigenesis associated with the p53 tumour suppressor gene.

The p53 gene is contained within 16-20 kb of cellular DNA located on the short arm of human chromosome 17 at position 17p13.1. This gene encodes a 393-amino-acid nuclear phosphoprotein involved in the regulation of cell proliferation. Current evidence suggests that loss of normal p53 function is associated with cell transformation in vitro and development of neoplasms in vivo. More than 50% of human malignancies of epithelial, mesenchymal, haematopoietic, lymphoid, and central nervous system origin analysed thus far, were shown to contain an altered p53 gene. The oncoproteins derived from several tumour viruses, including the SV40 large T antigen, the adenovirus E1B protein and papillomavirus E6 protein, as well as specific cellular gene products, e.g. murine double minute-2 (MDM2), were found to bind to the wild-type p53 protein and presumably lead to inactivation of this gene product. Therefore, the inactivation of p53 tumour suppressor gene is currently regarded as an almost universal step in the development of human cancers. The current data on p53-associated tumourigenesis are briefly discussed in this minireview.

Expression of p53 protein related to the presence of human papillomavirus (HPV) DNA in genital carcinomas and precancer lesions.

The E6 protein of the high-risk human papillomavirus (HPV) types 16 and 18 is capable of complexing with the wild-type p53 tumor suppressor gene product, leading to loss of the normal p53 function as an anti-oncogene, whereas the low-risk HPV types 6 and 11 lack this binding property. The malignant potential of HPV 16 and 18 has been ascribed to this complexing of E6 with p53, which regularly leads to undetectable expression of the latter in HPV-positive lesions. To assess the role of p53 in HPV-associated genital carcinogenesis, the expression of p53 protein was studied immunohistochemically in 22 genital carcinomas and precancer lesions; 8 vulvar carcinomas, 1 VIN (vulvar intraepithelial neoplasia), 5 cervical carcinomas and 8 CIN (cervical intraepithelial neoplasia) using monoclonal antibody PAb 1801. Presence of HPV was demonstrated by PCR using HPV consensus primers, and amplified HPV-DNA was digested with the restriction enzymes giving distinct patterns for various HPV-types in gel electrophoresis. HPV-typing was confirmed by in situ hybridization with biotinylated DNA probes. Altogether, 17 of the 22 specimens (77%) showed p53 expression: 67% of the precancer lesions and 83% of carcinomas. Expression was more frequent (89%) in the vulvar than (70%) in cervical lesions. Using PCR,HPV DNA was detected in 19/22(86%) of the samples. The following HPV types were identified: HPV 6 (2 samples), HPV 11 (3 cases), HPV 16 (5 cases), HPV 33 (3 cases), and 6 contained unidentified HPV types. All HPV DNA-negative specimens showed p53 expression. Of the 19 HPV DNA-positive lesions, 5 were p53-negative, three of these being HPV 16 positive CIN lesions. The remaining two HPV 16 lesions were invasive carcinomas with a weak p53 expression. HPV 6 and 11-positive lesions showed a weak p53 expression more frequently than HPV-negative cases and HPV 33 lesions. The results indicate that p53 expression is detectable, but it is less frequent and less intense in HPV DNA-positive genital precancer lesions and carcinomas (particularly those with HPV 16 DNA) as compared with HPV DNA-negative lesions.

Detection of human papillomavirus infections in the male sexual partners of women attending an STD clinic in Bologna.

A series of 65 male sexual partners of 65 women attending an STD clinic in Bologna, Italy for examination and treatment of genital human papillomavirus (HPV)-infections during 1990-1991, were examined using peniscopy and surgical biopsy, the latter being analysed by light microscopy, in situ hybridization (ISH) and polymerase chain reaction (PCR) for HPV DNA. A detailed medical and sexual history was recorded from all men. Of the 65 men, 17 (26.2%) gave a history of a previous STD. The male partners with previous genital condylomata (14, 21.5% of men) were significantly associated with the detection of HPV DNA in the current lesions; 21.4% (3 of 14) and 10.2% (5 of 51) in those with and without previously treated condyloma, respectively. On colposcopy, 63 (96.9%) men presented with an abnormal pattern, the vast majority (49 of 65, 75.4%) showing an acetowhite lesion, and only 12 (18.5%) lesions being classified as condyloma acuminatum. HPV DNA was found, however, in only 4 of 12 (33.3%) condylomas by ISH and PCR, and in 4 of 49 (8.2%) and 6 of 49 (12.2%) acetowhite lesions by ISH and PCR, respectively. In a total of 41 (63%) patients, the biopsy was classified as non-HPV on light microscopy. HPV DNA detection rate was significantly higher in all morphologically HPV-suggestive lesions, compared with the non-HPV where ISH was invariably negative. PCR, however, disclosed HPV DNA in 4 of 41 (9.8%) cases. PIN (I or II) was present in 6 of 65 (9.2%) men.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical course of cervical human papillomavirus lesions in relation to coexistent cervical infections.

A prospective follow-up of 530 women with cervical human papillomavirus (HPV) infection was conducted from 1981 to the present (mean 62.9 months). The patients were examined by PAP smears and colposcopy with or without biopsies every sixth month. Endocervical swabs were taken for culture of cytomegalovirus (CMV), herpes simplex virus (HSV), and Chlamydia trachomatis at each visit. During the follow-up period, 179 of the 530 patients (33.8%) had cervical infection and 351 (66.2%) had no coexistent cervicitis. On average, the patients with coexistent cervicitis were younger than those without cervicitis (32 +/- 7.2 years and 37.1 +/- 11.4 years, respectively; P less than 0.0001). C. Trachomatis was isolated from 95 of the 530 women (17.9%), and 19 of the patients had chlamydial cervicitis twice. Cytomegalovirus was isolated from 27 (5.1%) women, 2 of whom also had HSV, and 12 patients had a chlamydial infection. Herpes simplex virus was isolated from 11 (2.1%) women, including 2 patients with coexistent CMV infection. A total of 60 (1.3%) women had nonspecific cervicitis. Of the HPV lesions without coexistent cervical infection, 56.7% regressed, 24.5% persisted, 16.5% progressed, and recurrence was found in 2.3%. The corresponding figures for HPV lesions with coexistent cervicitis were as follows: 66.5%, 22.9%, 9.5%, and 1.1%, respectively. Coexistent active cervical infections had no influence on the clinical course of HPV lesions.

Immunohistochemistry, in situ hybridization and polymerase chain reaction (PCR) in detecting c-myc expression in human malignancies.

The assessment of oncogene expression at cellular level is important in understanding the role of those genes in carcinogenesis. Using in situ hybridization and immunohistochemistry, the expression of oncogenes can be visualized in topographic relation to tissue morphology. In the present study, c-myc overexpression was studied in ten carcinomas of different origin (6 mammary adenocarcinomas, 2 vulvar and 2 bronchial squamous cell carcinomas) by in situ hybridization (ISH) with 35S-labeled RNA probes and by immunohistochemistry (IHC). DNA amplification and transcription of c-myc oncogene were also studied with polymerase chain reaction (PCR) using beta-globin as an intrinsic standard for DNA amplification. The effect of formalin fixation of c-myc expression was simultaneously studied. Half of the tumours (5/10) demonstrated c-myc mRNA overexpression by ISH performed on frozen sections and two of the samples were shown to over-express c-myc protein by IHC. Only two samples fixed in formalin showed positive signals for c-myc mRNA. None of the biopsies showed DNA amplifications either with ISH or PCR. The present results suggest that ISH with RNA probes is a useful method for detecting the transcription of activated oncogenes in malignant tissues, especially when applied on frozen sections. The results also indicate that in some cases, c-myc gene may be adequately transcribed to mRNA but the latter is not translated to the appropriate oncoprotein.

A simple method for isolation of DNA from formalin-fixed paraffin-embedded samples for PCR.

A simple method of processing formalin-fixed, paraffin-embedded tissue sections for DNA amplification by polymerase chain reaction (PCR) is described. In this procedure, deparaffinized sections are readily subjected to DNA isolation simply by boiling and the released DNA can be directly employed for PCR. The method allows analysis of single-copy genes or viral sequences at least up to 300 base pairs long in one working day. This method is particularly useful in analysing retrospective materials when the simplicity and low cost of the assay are preferable. Furthermore, the simplicity of the procedure reduces the risk of contamination.