Methylome-wide analysis of milk somatic cells upon subclinical mastitis in dairy cattle.

Better understanding of the molecular mechanisms behind bovine mastitis is fundamental for improving the management of this disease, which continues to be of major concern for the dairy industry, especially in its subclinical form. Disease severity and progression depend on numerous aspects, such as livestock genetics, and the interaction between the causative agent, the host, and the environment. In this context, epigenetic mechanisms have proven to have a role in controlling the response of the animal to inflammation. Therefore, in this study we aimed to explore genome-wide DNA methylation of milk somatic cells (SC) in healthy cows (n = 15) and cows affected by naturally occurring subclinical mastitis by Streptococcus agalactiae (n = 12) and Prototheca spp. (n = 11), to better understand the role of SC methylome in the host response to disease. Differentially methylated regions (DMR) were evaluated comparing: (1) Strep. agalactiae-infected versus healthy; (2) Prototheca-infected versus healthy, and (3) mastitis versus healthy and (4) Strep. agalactiae-infected versus Prototheca-infected. The functional analysis was performed at 2 levels. To begin with, we extracted differentially methylated genes (DMG) from promoter DMR, which were analyzed using the Cytoscape ClueGO plug-in. Coupled with this DMG-driven approach, all the genes associated with promoter-methylated regions were fed to the Pathifier algorithm. From the DMR analysis, we identified 1,081 hypermethylated and 361 hypomethylated promoter regions in Strep. agalactiae-infected animals, while 1,514 hypermethylated and 358 hypomethylated promoter regions were identified in Prototheca-infected animals, when compared with the healthy controls. When considering infected animals as a whole group (regardless of the pathogen), we found 1,576 hypermethylated and 460 hypomethylated promoter regions. Both pathogens were associated with methylation differences in genes involved in pathways related to meiosis, reproduction and tissue remodeling. Exploring the whole methylome, in subclinically infected cows we observed a strong deregulation of immune-related pathways, such as nuclear factor kB and toll-like receptors signaling pathways, and of energy-related pathways such as the tricarboxylic acid cycle and unsaturated fatty acid biosynthesis. In conclusion, no evident pathogen-specific SC methylome signature was detected in the present study. Overall, we observed a clear regulation of host immune response driven by DNA methylation upon subclinical mastitis. Further studies on a larger cohort of animals are needed to validate our results and to possibly identify a unique SC methylome that signifies pathogen-specific alterations.

Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between High- and Low-motile sperm populations.

BACKGROUND: Small RNAs present in bovine ejaculate can be linked to sperm abnormalities and fertility disorders. At present, quality parameters routinely used in semen evaluation are not fully reliable to predict bull fertility. In order to provide additional quality measurements for cryopreserved semen used for breeding, a method based on deep sequencing of sperm microRNA (miRNA) and Piwi-interacting RNA (piRNA) from individual bulls was developed. To validate our method, two populations of spermatozoa isolated from high and low motile fractions separated by Percoll were sequenced, and their small RNAs content characterized. RESULTS: Sperm cells from frozen thawed semen samples of 4 bulls were successfully separated in two fractions. We identified 83 miRNAs and 79 putative piRNAs clusters that were differentially expressed in both fractions. Gene pathways targeted by 40 known differentially expressed miRNAs were related to apoptosis. Dysregulation of miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184 and miR-20a-5p was found to target three pathways (PTEN, PI3K/AKT and STAT). CONCLUSIONS: Small RNAs sequencing data obtained from single bulls are consistent with previous findings. Specific miRNAs are differentially represented in low versus high motile sperm, suggesting an alteration of cell functions and increased germ cell apoptosis in the low motile fraction.

Genomic and transcriptomic comparison between Staphylococcus aureus strains associated with high and low within herd prevalence of intra-mammary infection.

BACKGROUND: Staphylococcus aureus (Staph. aureus) is one of the major pathogens causing mastitis in dairy ruminants worldwide. The chronic nature of Staph. aureus infection enhances the contagiousness risk and diffusion in herds. In order to identify the factors involved in intra-mammary infection (IMI) and diffusion in dairy cows, we investigated the molecular characteristics of two groups of Staph. aureus strains belonging to ST8 and ST398, differing in clinical properties, through comparison of whole genome and whole transcriptome sequencing. RESULTS: The two groups of strains, one originated from high IMI prevalence herds and the other from low IMI prevalence herds, present a peculiar set of genes and polymorphisms related to phenotypic features, such as bacterial invasion of mammary epithelial cells and host adaptation. Transcriptomic analysis supports the high propensity of ST8 strain to chronicity of infection and to a higher potential cytotoxicity. CONCLUSIONS: Our data are consistent with the invasiveness and host adaptation feature for the strains GTB/ST8 associated to high within-herd prevalence of mastitis. Variation in genes coding for surface exposed proteins and those associated to virulence and defence could constitute good targets for further research.

In vitro algicidal effect of guanidine on Prototheca zopfii genotype 2 strains isolated from clinical and subclinical bovine mastitis.

Prototheca species have increasingly been reported to be opportunistic pathogens that cause mastitis in dairy herds, and it poses an emergent problem because at present, there are no effective therapies for the treatment of protothecal mastitis. This study investigated the in vitro algicidal effect of guanidine on 75 Prototheca zopfii genotype 2 strains isolated from 75 cases of clinical and subclinical bovine mastitis. All strains were susceptible to guanidine in vitro with minimal algaecide concentrations ranging from 0·001 to 0·035%. Guanidine is known to have a high microbicidal effect and is considered to be a new generation microbicidal compound. It is not toxic to human mucous membranes and conjunctivas at low concentrations and has been used as a disinfectant in swimming pools and as an antiseptic for human wounds. The algicidal action of guanidine at low concentrations indicates that it could be an alternative disinfectant or antiseptic for cleaning of the dairy environment and milking equipment, in pre- and postdipping solutions, in the chemical dry therapy of bovine teats and even in the intramammary therapy of P. zopfii infections. This is the first report of the in vitro algicidal effect of guanidine on P. zopfii strains of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca zopfii genotype 2 is an opportunistic pathogen of bovine mastitis. To date, no effective therapies against protothecal mastitis have been developed. The in vitro algicidal effect of guanidine on 75 P. zopfii genotype 2 strains isolated from cows revealed that all of the isolates were susceptible to the compound at low concentrations, which indicates that guanidine may be used as an antiseptic/disinfectant for dairy milking equipment, in pre- and postdipping solutions, and as a chemical dry therapy or an intramammary therapy. This study describes the in vitro algicidal effect of guanidine on P. zopfii for the first time.

Molecular typing and differences in biofilm formation and antibiotic susceptibilities among Prototheca strains isolated in Italy and Brazil.

Bovine mastitis caused by Prototheca is a serious and complex problem that accounts for high economic losses in the dairy industry. The main objective of this study was to identify and characterize at genetic level different Prototheca strains and provide the most complete data about protothecal antibiotic resistance. The study involves 46 isolates from Italian (13 strains) and Brazilian (33 strains) mastitic milk. These strains were identified by multiplex PCR and single strand conformation polymorphism analysis and characterized by randomly amplified polymorphic DNA (RAPD)-PCR. Moreover, biofilm production and antibiotic susceptibility were evaluated. Forty-two strains resulted as Prototheca zopfii genotype 2, whereas 4 isolates could belong to a potential new Prototheca species. The RAPD-PCR, performed with 3 primers (M13, OPA-4, and OPA-18), showed a notable heterogeneity among isolates and grouped the strains according to the species and geographical origin. Biofilm production was species-dependent and P. zopfii genotype 2 strains were classified as strong biofilm producers. In vitro antibiotic susceptibility tests indicated that Prototheca strains were susceptible to antibacterial drugs belonging to aminoglycosides group; the highest activity against Prototheca strains was observed in the case of colistin sulfate, gentamicin, and netilmicin (100% of susceptible strains). It is interesting to note that all the Italian P. zopfii genotype 2 strains showed lower minimum inhibitory concentration values than the Brazilian ones. Nisin showed more efficacy than lysozyme and potassium sorbate, inhibiting 31% of the strains. Results obtained in this study confirmed that RAPD-PCR is a rapid, inexpensive, and highly discriminating tool for Prototheca strains characterization and could give a good scientific contribution for better understanding the protothecal mastitis in dairy herd.

Genomic characteristics of Staphylococcus aureus strains associated with high within-herd prevalence of intramammary infections in dairy cows.

Staphylococcus aureus is one of the most important causes of mastitis in dairy cattle. Based on previous research, Staph. aureus genotypes with different pathogenic and contagious properties can cause intramammary infection (IMI) and coexist in the same herd. Our study aimed to compare Staph. aureus strains from herds that differed in IMI prevalence using different molecular approaches such as ribosomal spacer (RS)-PCR, multilocus sequence typing (MLST), spa typing, ribotyping, pulsed-field gel electrophoresis (PFGE), and multiplex PCR. For this purpose, 31 dairy herds with Staph. aureus IMI were selected, and 16 of these were chosen for a comparison study: the 8 high-prevalence (HP) herds had Staph. aureus IMI prevalence >28% and the 8 low-prevalence (LP) herds had an IMI prevalence <4%. A total of 650 isolates of Staph. aureus from mammary quarters of all positive cows were genotyped with RS-PCR, a technique based on amplification of a portion of the intergenic spacer 16S-23S rRNA, and a subset of 54 strains was also analyzed by multiplex PCR, ribotyping, PFGE, MLST, and spa typing. The RS-PCR analysis revealed 12 different profiles. Staphylococcus aureus strains isolated from 5 out of 8 HP herds showed a profile identical to the genotype B (GTB), described in previous studies as being strongly associated with high within-herd prevalence of Staph. aureus mastitis and the presence of the genes coding for enterotoxins sea, sed, and sej, a long x-region of spa gene, and 3 lukE fragments. Moreover, all strains isolated in the HP herds possessed genes coding for staphylococcal enterotoxins. In LP herds, a limited number of strains of 6 genotypes, different from those isolated in HP herds, were identified and GTB was not found. Within these genotypes, 4 strains were positive for the mecA gene. Preliminary results and comparison with other genotyping methods confirmed that genotyping by RS-PCR is an accurate, rapid, and inexpensive tool for future field studies on Staph. aureus mastitis strains and generates clinically relevant results.

Simultaneous identification by multiplex PCR of major Prototheca spp. isolated from bovine and buffalo intramammary infection and bulk tank.

UNLABELLED: Bovine mastitis caused by Prototheca spp. infection is increasing worldwide, therefore becoming more relevant to the dairy industry. Almost all Prototheca isolates from bovine mammary protothecosis came from P. zopfii genotype 2, with a lower prevalence of infection due to P. blaschkeae and rarely to P. wickerhamii. In this study, we report the development of two multiplex PCR assays able to discriminate among the three species responsible for bovine intramammary infection (IMI). Our assay is based on the specific amplification of new DNA target from mitochondria and chloroplasts partial sequences, of different Prototheca isolates. Both methods were set up using reference strains belonging to all Prototheca species and validated by the analysis of 93 isolates from bovine and buffalo IMI and bulk tank milk samples. The investigation involves 70 isolates from North, 13 from Central and 10 from South Italian regions. Isolates from bovine were most commonly identified as P. zopfii genotype 2, and only in one case as P. blaschkeae, whereas isolates from buffaloes belonged both to P. zopfii genotype 2 and P. wickerhamii. These findings proved the suitability of our multiplex PCRs as a rapid test to discriminate among pathogenic Prototheca strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports PCR assays based on novel Prototheca spp. mitochondrial and chloroplastic target sequences. The multiplex PCR protocol described in this study is useful for rapid simultaneous detection of P. zopfii, P. wickerhamii and P. blaschkeae.

Comparison between indicine and taurine cattle DNA methylation reveals epigenetic variation associated to differences in morphological adaptive traits.

Indicine and taurine subspecies present distinct morphological traits as a consequence of environmental adaptation and artificial selection. Although the two subspecies have been characterized and compared at genome-wide level and at specific loci, their epigenetic diversity has not yet been explored. In this work, Reduced Representation Bisulphite Sequencing (RRBS) profiling of the taurine Angus (A) and indicine Nellore (N) cattle breeds was applied to identify methylation differences between the two subspecies. Genotyping by sequencing (GBS) of the same animals was performed to detect single nucleotide polymorphisms (SNPs) at cytosines in CpG dinucleotides and remove them from the differential methylation analysis. A total of 660,845 methylated cytosines were identified within the CpG context (CpGs) across the 10 animals sequenced (5 N and 5 A). A total of 25,765 of these were differentially methylated (DMCs). Most DMCs clustered in CpG stretches nearby genes involved in cellular and anatomical structure morphogenesis. Also, sequences flanking DMC were enriched in SNPs compared to all other CpGs, either methylated or unmethylated in the two subspecies. Our data suggest a contribution of epigenetics to the regulation and divergence of anatomical morphogenesis in the two subspecies relevant for cattle evolution and sub-species differentiation and adaptation.

In vivo dosimetry with radiochromic films in low-voltage intraoperative radiotherapy of the breast.

PURPOSE: EBT2 radiochromic films were studied and used for in vivo dosimetry in targeted intraoperative radiotherapy (TARGIT), a technique in which the Intrabeam system (Carl Zeiss, Oberkochen, Germany) is used to perform intraoperative partial breast irradiation with x-rays of 50 kV(p). METHODS: The energy of the radiation emitted by the Intrabeam with the different spherical applicators, under 1 and 2 cm of solid water, and under the tungsten impregnated rubber used for shielding of the heart in TARGIT of the breast, was characterized with measurements of half-value layer (HVL). The stability of response of EBT2 was verified inside this range of energies. EBT2 films were calibrated using the red and green channels of the absorption spectrum in the 0-20 Gy dose range delivered by the Intrabeam x-rays. The dependence of film response on temperature during irradiation was measured. For in vivo dosimetry, pieces of radiochromic films wrapped in sterile envelopes were inserted after breast conserving surgery and before TARGIT into the excision cavity, on the skin and on the shielded pectoralis fascia for treatments of the left breast. RESULTS: HVLs of the Intrabeam in TARGIT of the breast correspond to effective energies of 20.7-36.3 keV. The response of EBT2 was constant inside this range of energies. We measured the dose to the target tissue and to organs at risk in 23 patients and obtained an average dose of 13.52 ± 1.21 Gy to the target tissue. Dose to the skin in close proximity to the applicator was 2.22 ± 0.97 Gy, 0.29 ± 0.17 Gy at 5-10 cm from the applicator, and 0.08 ± 0.07 Gy at more than 10 cm from the applicator. Dose to the pectoral muscle for left breast treatment was 0.57 ± 0.23 Gy. CONCLUSIONS: Our results show that EBT2 films are accurate at the beam energies, dose range, and irradiation temperature found in TARGIT and that in vivo dosimetry in TARGIT with EBT2 films wrapped in sterile envelopes is a feasible procedure. Measured dose to the organs at risk indicates that the technique is safe from side effects to the skin and the heart.

Toll-like receptor 4 genetic diversity among pig populations.

The transmembrane glycoprotein encoded by the Toll-like receptor 4 gene (TLR4) acts as the transducing subunit of the lipopolysaccharide receptor complex of mammals, which is a major sensor of infections by Gram-negative bacteria. As variation in TLR4 may alter host immune response to lipopolysaccharide, the association between TLR4 polymorphisms and immune traits of the respiratory and gut systems has important implications for livestock. Here, a sequence dataset from 259 animals belonging to commercial and traditional European pig populations, consisting of 4305 bp of TLR4, including the full transcribed region, a portion of intron 2 and the putative promoter region, was used to explore genetic variation segregating at the TLR4 locus. We identified 34 single nucleotide polymorphisms, 17 in the coding sequence and 17 in the non-coding region. Five non-synonymous mutations clustered within, or in close proximity to, the hypervariable domain of exon 3. In agreement with studies in other mammals, a major exon 3 haplotype segregated at high frequency in the whole sample of 259 pigs, while variants carrying non-synonymous substitutions showed frequencies ranging between 0.6% and 8.7%. Although results on exon 3 provided suggestive evidence for purifying selection occurring at the porcine TLR4 gene, the analysis of both coding and non-coding regions highlighted the fact that demographic factors strongly influence the tests of departure from neutrality. The phylogenetic analysis of TLR4 identified three clusters of variation (ancestral, Asian, European), supporting the evidence of Asian introgression in European main breeds and the well documented history of pig breed domestication previously identified by mtDNA analysis.

Evaluation of the effects of different diets on microbiome diversity and fatty acid composition of rumen liquor in dairy goat.

Fat supplementation plays an important role in defining milk fatty acids (FA) composition of ruminant products. The use of sources rich in linoleic and α-linolenic acid favors the accumulation of conjugated linoleic acids isomers, increasing the healthy properties of milk. Ruminal microbiota plays a pivotal role in defining milk FA composition, and its profile is affected by diet composition. The aim of this study was to investigate the responses of rumen FA production and microbial structure to hemp or linseed supplementation in diets of dairy goats. Ruminal microbiota composition was determined by 16S amplicon sequencing, whereas FA composition was obtained by gas-chromatography technique. In all, 18 pluriparous Alpine goats fed the same pre-treatment diet for 40±7 days were, then, arranged to three dietary treatments consisting of control, linseed and hemp seeds supplemented diets. Independently from sampling time and diets, bacterial community of ruminal fluid was dominated by Bacteroidetes (about 61.2%) and Firmicutes (24.2%) with a high abundance of Prevotellaceae (41.0%) and Veillonellaceae (9.4%) and a low presence of Ruminococcaceae (5.0%) and Lachnospiraceae (4.3%). Linseed supplementation affected ruminal bacteria population, with a significant reduction of biodiversity; in particular, relative abundance of Prevotella was reduced (-12.0%), whereas that of Succinivibrio and Fibrobacter was increased (+50.0% and +75.0%, respectively). No statistically significant differences were found among the average relative abundance of archaeal genera between each dietary group. Moreover, the addition of linseed and hemp seed induced significant changes in FA concentration in the rumen, as a consequence of shift from C18 : 2n-6 to C18 : 3n-3 biohydrogenation pathway. Furthermore, dimethylacetal composition was affected by fat supplementation, as consequence of ruminal bacteria population modification. Finally, the association study between the rumen FA profile and the bacterial microbiome revealed that Fibrobacteriaceae is the bacterial family showing the highest and significant correlation with FA involved in the biohydrogenation pathway of C18 : 3n-3.

Normal tissue complication probability models for severe acute radiological lung injury after radiotherapy for lung cancer.

PURPOSE: To derive Normal Tissue Complication Probability (NTCP) models for severe patterns of early radiological radiation-induced lung injury (RRLI) in patients treated with radiotherapy (RT) for lung tumors. Second, derive threshold doses and optimal doses for prediction of RRLI to be used in differential diagnosis of tumor recurrence from RRLI during follow-up. METHODS AND MATERIALS: Lyman-EUD (LEUD), Logit-EUD (LogEUD), relative seriality (RS) and critical volume (CV) NTCP models, with DVH corrected for fraction size, were used to model the presence of severe early RRLI in follow-up CTs. The models parameters, including α/ß, were determined by fitting data from forty-five patients treated with IMRT for lung cancer. Models were assessed using Akaike information criterion (AIC) and area under receiver operating characteristic curve (AUC). Threshold doses for risk of RRLI and doses corresponding to the optimal point of the receiver operating characteristic (ROC) curve were determined. RESULTS: The α/ßs obtained with different models were 2.7-3.2 Gy. The thresholds and optimal doses curves were EUDs of 3.2-7.8 Gy and 15.2-18.1 Gy with LEUD, LogEUD and RS models, and µd of 0.013 and 0.071 with the CV model. NTCP models had AUCs significantly higher than 0.5. Occurrence and severity of RRLI were correlated with patients' values of EUD and µd. CONCLUSIONS: The models and dose levels derived can be used in differential diagnosis of tumor recurrence from RRLI in patients treated with RT. Cross validation is needed to prove prediction performance of the model outside the dataset from which it was derived.

Cytogenetics of multiple endocrine neoplasia syndromes. I. Two different, unique clonal chromosome changes in a medullary thyroid carcinoma and in a C-cell thyroid hyperplasia.

In short-term cultures of tumor tissue from a medullary thyroid carcinoma (MTC), we found a large clone of cells with a balanced translocation t(9;12)(p24;q22). A large clone with a balanced translocation t(10;16)(p11;q24) was also found in cultures from a C-cell thyroid hyperplasia. No clearcut evidence for chromosome instability was observed in the lymphocytes of the two patients. The mother of the first patient died of MTC; two relatives of the second patient had MTC and one of them had pheochromocytoma. These findings classify the two subjects as MEN 2A patients with different phenotypic expression but with the same type of chromosomal abnormality.

Chromosome abnormalities in a case of pituitary adenoma.

Chromosome analysis of primary cultures from an ACTH-secreting pituitary adenoma revealed the presence of one clone of cells with deletion of 18p and some random structural and numerical abnormalities.

Cytogenetic studies in venous tissue from patients with varicose veins.

Cytogenetic investigation of primary cell cultures from fragments of varicose veins of seven patients with familial varicosity and seven patients with the sporadic type revealed the presence of metaphases with structural abnormalities, clonal trisomies of chromosomes 7, 12, and 18, and monosomy of chromosome 14 only in cases with the familial type, while the sporadic cases had no similar chromosome aberrations. The immunophenotypical results are consistent with fibroblast lineage of the cultured cells. These results suggest that karyotypic variations in familial varicose vein tissue cultures could in some way be associated either with the genotypic constitution responsible for the familial type or a longer duration of disease on average than those with sporadic varicosities.

Cytogenetics of multiple endocrine neoplasia syndrome. II. Chromosome abnormalities in an insulinoma and a glucagonoma from two subjects with MEN1.

Cytogenetic analysis of two pancreatic islet tumors, an insulinoma and a glucagonoma was ascertained in two subjects with multiple endocrine neoplasia type 1 (MEN1). The insulinoma had a modal peak at 84 chromosomes. Most cells were pseudotetraploid, and in all cells the normal chromosomes were represented in varied numbers, i.e., from 1 to 7 copies. The tumor had 5 characteristic and consistent marker chromosomes which were identified as deletions of chromosomes 1, 2, 7, 16, and 17. All metaphases had several double minute chromosomes (dmin) of variable size and possible intermediate structures between dmin and homogeneously staining chromosomal regions. The glucagonoma had a nearly equal proportion of normal metaphases and metaphases with structural and numerical abnormalities with no consistent trend.

A search for double minute chromosomes in cultured lymphocytes from different types of tumors.

Double minutes are considered the products of DNA amplification and are rare in normal human cells. They have been observed in cultured lymphocytes in selected samples of human populations as one of the characteristics of the so-called rogue cells. We scored 9500 metaphases of cultured lymphocytes from 65 subjects with a variety of heredity and sporadic tumors and from 30 healthy subjects. The 15 cells with double minutes were found in subjects with multiple endocrine neoplasia type 1 (14 cases) and with familial adenomatous polyposis (1 case). Only one rogue cell was found among the 15 cells with double minutes.

Ingested soil as a source of 137Cs to ruminants.

Ingestion of soil has been identified as a potentially important source of radionuclides, particularly to grazing ruminants. Soil artificially contaminated with radiocaesium was administered orally each day for 33 days to Bergamasca sheep and the transfer to milk measured. Two soil types were used, with a clay content of 11% and 16%, respectively. Transfer coefficient (fm) values to sheep milk, calculated at the equilibrium (period from 7th to 21st day after soil administration), were 3.72 x 10(-4) days l-1 (S.D. = 7.2 x 10(-5) days l-1) and 6.11 x 10(-4) days l-1 (S.D. = 9.1 x 10(-5) days l-1), respectively for the two soils. These low values indicate that radiocaesium is not removed from the binding sites on the clay minerals in the gut of the sheep.

[Anti-HCV antibodies in chronically dialyzed uremic patients. 1-year follow-up study]. / Anticorpi anti-HCV in pazienti uremici cronici dializzati. Studio ad un anno di distanza.

The sera of 109 patients undergoing chronic haemodialysis treatment were reexamined after one year in order to assess changes in the anti-HVC antibody pattern in the intervening period, June 1992-June 1993. Using the ELISA II generation test, positive cases were found to have risen from 57 to 63 (from 52.3% to 57.8%); the Riba II test showed 60 positive cases (previously 52) with 3 undetermined (previously 5). The incidence of biochemical indicators of necrosis and/or cholestasis, negative in HCV patients, also presents a particular positivity (44%) in the presence of four antibody fractions. These data confirm the importance of serial determinations in anti-HCV antibody time, even if they do not correlate directly with the presence of the virus in the circulation and hence with its infecting capacity, the marker for which should be sought in the polymerase chain reaction.

Concurrent cytogenetic and molecular investigations in uterine and ovarian neoplasms.

We studied a group of 24 uterine and ovarian neoplasms with the purpose to verify if any correlation could be established between chromosomal abnormalities, loss of heterozigosity (LOH) and microsatellite instability (MIN). Tumor specimens obtained from 24 women (12 affected by ovarian and 12 by uterine neoplasms) were split in two parts, one was used for short term cultures for cytogenetic investigation while from the second DNA was extracted for molecular studies. We studied 22 polymorphic loci from 19 chromosomes and compared the alleles observed in the tumor with those observed in the DNA obtained from peripheral blood. Extensive loss of heterozigosity was observed when total or partial chromosomal loss was observed in at least 50% of the examined cells; MIN did not correlate with any particular cytogenetic abnormality nor with LOH.