Concomitant carotid plaque development and brachial artery diameter enlargement: a retrospective, recall-based study in postmenopausal women.

BACKGROUND AND AIM: To verify if the carotid plaque development is concomitant to brachial artery diameter enlargement, in healthy postmenopausal women. METHODS AND RESULTS: This is a retrospective, recall study. We enrolled 40 postmenopausal women, selected from a database for the period 2000-2008, not affected by subclinical carotid atherosclerosis and without risk factors for cardiovascular disease. At the recall visit, carotid and brachial duplex scan was again obtained. The incidence of plaque was 30% after a mean follow-up period of 60 months. There were no differences in baseline characteristics between subjects developing carotid atherosclerosis and subjects who did not, except for the brachial diameter change, follow-up and heart rate. The logistic-regression analysis confirmed that only brachial diameter change resulted to be correlated with the development of carotid atherosclerosis. CONCLUSION: Brachial artery diameter increase is concomitant to carotid plaque development. Vascular enlargement could not be a focal change but a systemic process associated with atherosclerotic plaque development. Brachial diameter could be a tool with a predictive significance.

Brachial artery diameter measurement: a tool to simplify non-invasive vascular assessment.

AIM: The mechanisms of vascular remodeling have attracted great interest since it is a phenomenon related to cardiovascular diseases. We would like to examine studies that contributed to clarify the remodeling mechanisms, to explore the different faces of atherosclerosis process. DATA SYNTHESIS: A number of invasive and non-invasive vascular assessment methods were developed, to detect the early sign of atherosclerosis. It became clear that the invasive tests were not applicable to large-scale studies. Consequently, a non-invasive test was developed. Studies showed that the endothelial function evaluation is a predictor of future cardiac events in individuals at cardiovascular risk and in those with established disease. However, analyzing several works, an interesting concept emerged, i.e., the inverse relation between endothelium-dependent dilation and vessel size, since large vessel tend not to dilate significantly. This notion emphasized the role of basal diameter on vascular response. In particular, as brachial artery diameter is the measure on which FMD is based, it could add more information in clinical evaluation, simplifying the assessment. Several studies showed that morphological change of brachial artery is a better indicator of the extent of coronary disease rather than FMD. Other studies showed that brachial diameter has predictive significance in the stratification of cardiovascular risk. CONCLUSION: Brachial diameter is a useful and simple tool. It should be incorporated into the overall assessment of cardiovascular risk but further studies are warranted to determine the final place of brachial diameter assessment in routine clinical setting.

Large brachial artery diameter and diabetes in post-menopausal women.

BACKGROUND AND AIM: Vascular remodelling is one of the possible compensatory mechanisms in response to artery wall injury. It was demonstrated that post-menopausal women with carotid atherosclerosis had a larger brachial artery diameter (BAD) than women without carotid plaques. Therefore, it is possible to hypothesise that artery enlargement could be a marker of early atherosclerosis. To investigate the eventual association between carotid and brachial artery diameter and disease affecting the vascular wall, we performed a case-control study in post-menopausal women with or without type II diabetes mellitus. METHODS AND RESULTS: We enrolled 28 cases (with diabetes) and 56 controls (without diabetes) matched for age and carotid atherosclerosis presence and severity. On the t-test, women with diabetes showed significantly larger brachial and common carotid artery diameters and, as expected, higher plasma glucose level and homeostasis model assessment (HOMA) than women without diabetes. On the univariate analysis, only plasma glucose level results correlated to BAD in the whole sample. Multivariate analysis confirmed that diabetes was a good predictor of brachial and carotid artery diameter, while age, systolic blood pressure and triglycerides were correlated only to the carotid diameter. CONCLUSIONS: Our data confirm that vascular remodelling is a systemic process occurring in conditions related to atherosclerosis, such as type II diabetes. Indeed, artery diameter could be a marker of early response of vessel wall to injury.

Artery remodeling and abdominal adiposity in nonobese postmenopausal women.

BACKGROUND/OBJECTIVES: We aimed to assess, in nonobese postmenopausal women, whether markers of central adiposity, especially waist-to-hip ratio (WHR), would be associated with vascular remodeling. SUBJECT/METHODS: We enrolled 263 postmenopausal nonobese women without metabolic syndrome or diabetes. The strongest anthropometric measure related to brachial artery diameter (BAD) was WHR. Therefore, we divided the population in tertiles according to WHR values. Women in third tertiles were older, with higher body mass index, had worse lipid profile and a higher BAD than women in the first and second tertiles. RESULTS: An analysis of covariance confirmed that BAD was increased with increasing tertiles after correction for confounding variables. CONCLUSION: BAD, a surrogate measure of cardiovascular disease, was correlated to WHR in nonobese population; therefore, nonobese women with high WHR should be carefully considered because of a possible worse cardiovascular risk profile.

Hyperhomocysteinemia recurrence in levodopa-treated Parkinson's disease patients.

BACKGROUND: Patients with Parkinson's disease (PD) and chronically treated with L-DOPA exhibit, in a percentage of 10-30%, supra-physiological levels of plasma total homocysteinemia (tHcy). In this study, we have investigated, in a group of hyper-homocysteinemic PD patients, the time of hyper-tHcy recurrence after discontinuation of 1-month folate supplementation given to normalize plasma tHcy levels. METHODS: Plasma tHcy, cobalamin and folate were assayed before and after 1-month folate supplementation (5 mg/day), and after 2 and 4 months after folate discontinuation in 29 PD patients (16M/13F, mean age 69.4 +/- 6.9 years) stabilized on a mean L-DOPA dose of 509.4 +/- 312.1 mg/day. RESULTS: After folate supplementation, plasma tHcy levels fell within the normal range in all patients. At the 2-month control after folate discontinuation, plasma tHcy remained within physiological values in 25 out of 29 patients. Conversely, 4 months after folate discontinuation, all patients exhibited hyper-tHcy. CONCLUSIONS: One-month intake of 5 mg/day folate normalizes plasma tHcy levels in all hyper-homocysteinemic PD patients. Following folate discontinuation, hyper-tHcy recurs in all patients within 4 months. Knowledge of this time interval is useful to optimize pulses of folate therapy in hyper-homocysteinemic patients with PD.

Implications of metal exposure and liver function in Parkinsonian patients resident in the vicinities of ferroalloy plants.

Valcamonica is an Italian valley where ferro-manganese industries have been active for a century and where an increased prevalence of parkinsonism was observed. A group of 93 patients (65 from Valcamonica, 28 from the reference area of Brescia city) and 76 controls (52 from Valcamonica, 24 from Brescia) were screened for serum Cu, Zn, Fe, Mn in blood (MnB) and urine (MnU), transferrin, peroxides, alanine (ALT) and aspartate (AST) transaminases and direct bilirubin. Test results were compared among groups according to the residential area and related to the disease severity. Valcamonica patients had a serum-increase of Cu, as well as of AST/ALT ratio, and a serum-decrease of Zn and Fe compared with other subgroups of cases and controls. Cases and controls from Valcamonica had higher MnB and MnU levels compared to cases and controls from Brescia. After controlling for the duration of illness, the Unified Parkinson's Disease Rating Scale III domain correlated with serum Cu and AST/ALT ratio. Our results suggest the possibility that, in this area, a lifetime exposure to neurotoxicants and to Mn in particular, when accompanied to a subclinical liver dysfunction, may pose an increased risk for neurodegenerative disorders via metal metabolism (Cu, Zn, Fe) abnormalities.

Effect of MTHFR polymorphisms on hyperhomocysteinemia in levodopa-treated Parkinsonian patients.

High plasma homocysteine levels have been observed in Parkinson's disease (PD) patients treated with levodopa. In this study, we investigated the effects of C677T and A1298C MTHFR polymorphisms, in association with L-DOPA daily dose and vitamin status, on hyperhomocysteinemia development in PD patients. Plasma homocysteine and folate/vitamin B12 levels were assayed in 49 L-DOPA-treated PD patients, and compared with those of 86 healthy subjects. Genotyping for MTHFR polymorphisms was carried out by DG-DGGE. Homocysteine levels were significantly higher in patients than in controls (16.3 +/- 5.7 vs. 11.7 +/- 2.7 micromol/l, P < 0.01). No significant differences were found between patients and controls with regard to folate/vitamin B12 levels, and MTHFR allele distribution. The TT+AA genotype was significantly more frequent in PD patients than in controls (32.5% vs. 17.4%, P < 0.05), but not associated with an increased risk for PD (OR = 2.3, CI = 1.0-5.2). Further, patients carrier of this genotype exhibited a mild hyperhomocysteinemia (22.1 +/- 4.9 micromol/l), while a protective effect was observed in patients having the CC+AA genotype (11.2 +/- 1.6 micromol/l; OR = 0.19, CI = 0.06-0.59). Interestingly, homocysteine levels were also moderately increased in patients with CT heterozygous genotype, in the context of either AA or AC (14.5 +/- 3.6 micromol/l), in comparison to subjects with the CC + AA genotype. Finally, we did not find any significant association of combined C677T and A1298C MTHFR polymorphisms with an increased risk for hyperhomocysteinemia in PD patients. A better understanding of the role of homocysteine and MTHFR genotypes in PD is needed to reveal novel approaches for disease management.

Relation between serum uric acid and carotid intima-media thickness in healthy postmenopausal women.

OBJECTIVE: Serum uric acid (SUA) is associated with cardiovascular disease (CVD). However it is still disputed whether the relationship is mediated by other risk factors such as obesity, dyslipidaemia, hypertension and insulin resistance. We explored the association of the uric acid level with carotid intima-media thickness (IMT), a well known marker of CVD, in postmenopausal healthy women. METHODS: We consecutively enrolled postmenopausal women undergoing a screening for health evaluation. After an accurate clinical examination, and a biochemical evaluation, the enrolled subjects underwent B mode ultrasonography to assess common carotid intima media thickness. RESULTS: Among 234 women aged 45-70 years, the uric acid level is associated with carotid IMT independently of other prognostic factors (p=0.03). In particular, women in the highest tertiles of uric acid level have a greater IMT than women in the lowest tertile (p=0.007). CONCLUSIONS: Independently of other cardiovascular risk factors, SUA levels are associated with carotid IMT even in subjects without the metabolic syndrome. This confirms and expands the role of uric acid in the determinism of CVD. Prospective trials would be useful to evaluate interventions aimed at lowering the uric acid level.

Role of endogenous androgens on carotid atherosclerosis in non-obese postmenopausal women.

BACKGROUND: Recent randomized trials on hormone replacement therapy in postmenopausal women raised many doubts about their role in cardiovascular disease prevention. Therefore the role of other sex hormones needed to be investigated. In particular androgens seem to have a protective role on atherosclerosis. The present study was performed to assess the role of endogenous sex hormones on carotid atherosclerosis in postmenopausal women. METHODS AND RESULTS: We consecutively enrolled 101 postmenopausal women aged 45-75 (mean age 57.4) years referred to our University hospital menopausal health-screening clinic. The subjects underwent a medical history, a physical examination and biochemical analysis. Extracranial carotid arteries were assessed by ultrasound. Fifty percent of our sample had carotid plaques. On the multivariate logistic regression analysis age, glycaemia (positively) and testosterone (negatively) (P=0.02) were significantly correlated to carotid atherosclerosis. In non-obese subjects we found that participants in the third tertile had a significantly lower prevalence of carotid atherosclerosis (P=0.02) compared to those in the first tertile of testosterone. CONCLUSIONS: These results suggest a possible protective role of endogenous androgens at least on carotid atherosclerosis. Of course these preliminary results should be supported by prospective studies. Also the different role of these hormones on obese and non-obese subjects needs to be clarified.

[Metals and oxidative stress in Parkinson's disease from industrial areas with exposition to environmental toxins or metal pollution]. / Dismetabolismo dei metalli e stress ossidativo in pazienti con in Parkinson's disease di un'area industriale esposta ad inquinamento da metalli.

BACKGROUND: Parkinson's disease (PD) is characterized by a progressive degeneration of the nigrostriatal dopaminergic pathway resulting in movement disorders. PD is a complex disease, in which and environmental factors, as exposure to toxins or metals coul be involved. OBJECTIVE: To assess if serum metals (Cu, Fe, Zn), biological variables of their metabolism, total peroxides and antioxidants were abnormal in PD, in relation to environmental exposure. METHODS: We compared levels of serum copper, iron, zinc, ceruloplasmin and transferrin, peroxides, antioxidants (TRAP) in 65 PD patients coming from an Industrial zone highly exposed to metal pollution (Valcamonica) with measures from 28 PD patients from no metal pollution areas of the province of Brescia and 52 healthy controls coming from Valcamonica and 24 from the province of Brescia. RESULTS: PD patients had higher serum concentration of zinc than controls. Only in PD patients coming from Valcamonica levels of Cu were higher than in subjects coming from the province of Brescia. Moreover, In patients with PD levels of sieric Cu significantly correlated with score of the Unified Parkinson's Disease Rating Scale (UDPRS). CONCLUSIONS: Zinc seems to be higher in PD independently from the exposition to metal pollution. Perturbation of copper metabolism in PD seems to be related to exposition to environmental toxins or metal pollution and coul be involved in the progression of the disease itself.

Screening for C677T and A1298C MTHFR polymorphisms in patients with epilepsy and risk of hyperhomocysteinemia.

Hyperhomocysteinemia can result from decreased methylenetetrahydrofolate reductase (MTHFR) enzyme activity, owing to genetic polymorphisms andor inadequate folate intake. This study was aimed at investigating the prevalence of C677T and A1298C MTHFR polymorphisms, and their impact on hyperhomocysteinemia in 95 epileptic patients and 98 controls. Double gradient-denaturing gradient gel electrophoresis screening revealed that the frequency of T677 polymorphic allele was similar between cases and controls (46.3% vs 42.3%), whereas that of C1298 allele was significantly higher in patients (30.5% vs 19.4%, p < 0.05). Significant differences between the two groups were also found for the frequencies of genotypes AA1298 (46.3% in cases vs 67.3% in controls, p < 0.01) and AC1298 (46.3% in cases vs 26.6% in controls, p < 0.01). Other genotype frequencies did not show any statistically significant differences. Haplotype frequencies significantly differed between the two groups. The CT677/AC1298 diplotype was significantly more frequent in epileptic patients than in controls (32.6% vs 18.4%, p < 0.05). Patients treated with enzyme-inducing antiepileptic drugs, having this diplotype and concomitant low folate concentration (i.e., < 3.4 nmol/L), exhibited plasma homocysteine levels significantly higher than normal values (27.1 +/- 2.44 micromol/L, p < 0.001). This increase, however, was lower than that observed in folate-deficient patients with diplotype TT677/AA1298 (41.3 +/- 3.41 micromol/L, p < 0.001). Indeed, these two diplotypes could be regarded as risk factors for hyperhomocysteinemia. Conversely, we found that the CC677/AA1298 diplotype was significantly more frequent in controls (p < 0.01), suggesting a protective role. Our study suggests that both C677T and A1298C MTHFR polymorphisms should be examined when assessing genetic risk factors of hyperhomocysteinemia in epilepsy.

Awareness, treatment and control of hyperlipidaemia, hypertension and diabetes mellitus in a selected population of southern Italy.

The purpose of the present study was to assess the degree of awareness, treatment and control of hyperlipidaemia compared with hypertension and diabetes mellitus in a selected population of southern Italy. All participants to a cardiovascular disease prevention campaign examined between April 1994 and July 1995 were screened for hyperlipidaemia, hypertension and diabetes mellitus. Subjects received also ECG, echo-Doppler of carotid arteries and filled in a questionnaire concerning personal and familial cardiovascular diseases, smoking habit and drug consumption. Of the 742 participants, 327 were found to have hypertension, 73 to have diabetes mellitus, 287 to have mild hyperlipidaemia and 322 to have moderate-severe hyperlipidaemia. Among hypertensive subjects, 60.2% were aware of their condition, 53.5% were treated and 15.6% had their blood pressure controlled at the recommended level (< 140/90 mmHg). Among diabetic subjects, 76.7% were aware, 64.4% treated and 19.2% reached fasting blood glucose level of less than 7.77 mmol/l (140 mg/dl). Only 24.0% of subjects with mild hyperlipidaemia were aware of their condition. Of the subjects found to have moderate-severe hyperlipidaemia, 64.9% were aware, 32.3% were treated and 9.0% had plasma cholesterol and triglycerides concentration of less than 6.45 and 5.65 mmol/l (250 and 500 mg/dl), respectively (cutoffs chosen to separate mild from moderate-severe hyperlipidaemia). These results show that mild hyperlipidaemia is almost neglected whereas awareness of moderave-severe hyperlipidaemia is quite widespread and comparable to that of hypertension and diabetes mellitus. Prevalence of treatment and control of moderate-severe hyperlipidaemia is, however, much lower than that of hypertension and diabetes.

Expression of the chemokine RANTES by a human bronchial epithelial cell line. Modulation by cytokines and glucocorticoids.

The chemokine RANTES is a potent chemoattractant for eosinophils, lymphocytes, and monocytes, and has been detected recently in the epithelium of human airways mucosa. We have studied, therefore, the expression of RANTES mRNA and protein in the human bronchial epithelial cell line BEAS-2B. Using Northern blot analysis, RANTES mRNA was not detectable in unstimulated BEAS-2B cells. Incubation of cells with TNF-alpha or IFN-gamma induced the expression of RANTES mRNA and protein within 16 h. The combination of TNF-alpha and IFN-gamma displayed a marked synergism in inducing RANTES expression. Pretreatment of cells with the glucocorticoid budesonide (10(-10)-10(-7) M) for 24 h inhibited expression of RANTES mRNA and protein stimulated by either TNF-alpha or TNF-alpha plus IFN-gamma in a concentration- and time-dependent manner. Nonglucocorticoid steroids did not inhibit RANTES mRNA expression. Production of RANTES by epithelium could contribute to the mechanism of selective cellular recruitment occurring in the airways during inflammation, thus playing a relevant role in the pathogenesis of diseases such as asthma, rhinitis, and polyposis. The down-regulation of RANTES production by glucocorticoids in epithelial cells may contribute to the efficacy of these compounds in reducing cellular infiltration and, ultimately, to their anti-inflammatory properties.

Identification of an immunologically important hypervariable domain of major outer surface protein A of Borrelia burgdorferi.

The gene for the major outer surface protein A (OspA) from several clinically obtained strains of Borrelia burgdorferi, the cause of Lyme disease, has been cloned, sequenced, and expressed in Escherichia coli by using a T7-based expression system (J. J. Dunn, B. N. Lade, and A. G. Barbour, Protein Expr. Purif. 1:159-168, 1990). All of the OspAs have a single conserved tryptophan at residue 216 or, in some cases, 217; however, the region of the protein flanking the tryptophan is hypervariable, as determined by a moving-window population analysis of ospA from 15 European and North American isolates of B. burgdorferi. Epitope-mapping studies using chemically cleaved OspA and a TrpE-OspA fusion have indicated that this hypervariable region is important for immune recognition. Biophysical analysis, including fluorescence and circular dichroism spectroscopy, have indicated that the conserved tryptophan is buried in a hydrophobic environment. Polar amino acid side chains flanking the tryptophan are likely to be exposed to the hydrophilic solvent. The hypervariability of these solvent-exposed amino acid residues may contribute to the antigenic variation in OspA. To test this, we have performed site-directed mutagenesis to replace some of the potentially exposed amino acid side chains in the B31 protein with the analogous residues of a Borrelia garinii strain, K48. The altered proteins were then analyzed by Western blot (immunoblot) with monoclonal antibodies which bind OspA on the surface of the intact B31 spirochete. Our results indicate that specific amino acid changes near the tryptophan can abolish the reactivity of OspA to these monoclonal antibodies, which is an important consideration in the design of vaccines based on recombinant OspA.

Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-gamma plus TNF-alpha and inhibition by IL-4 and IL-13.

Production by endothelial cells of the regulated on activation normal T expressed and secreted chemokine (RANTES) has recently been evidenced during delayed-type hypersensitivity (DTH) reactions and may contribute to the local accumulation of macrophages and CD4+ memory T lymphocytes. To document the mechanism inducing RANTES production in this condition, we analyzed the effect of cytokines known to influence the formation of DTH granulomas. Little or no RANTES was produced after stimulation of HUVEC with IFN-gamma, IL-1 beta, or TNF-alpha. However, the combination TNF-alpha+IFN-gamma induced a strong RANTES production. In situ hybridization experiments with a RANTES probe showed that this synergy was also observed at the mRNA level and that the effect of the combination was mainly to increase the amount of RANTES mRNA per cell. The expression of the luciferase gene under the control of the RANTES gene regulatory elements was analyzed; TNF-alpha and the combination TNF-alpha+IFN-gamma activated the regulatory elements. Sequential treatment of HUVEC with TNF-alpha and IFN-gamma showed that IFN-gamma sensitized HUVEC to the stimulating effect of TNF-alpha. The production of RANTES induced by TNF-alpha+IFN-gamma was partly but significantly inhibited by the Th2-type cytokines IL-4 and IL-13. In contrast, IL-10 had no effect. These results indicate that the microenvironment of DTH granulomas, containing high levels of both TNF-alpha and IFN-gamma, may be responsible for RANTES production by perigranulomatous endothelial cells. Inhibition of this production by Th2-type cytokines may be a mechanism by which these cytokines interfere with the formation of DTH granulomas.

[Correlation of waist-to-hip circumference ratio and cardiovascular risk factors in a population of Southern Italy]. / Rapporto circonferenza addome/circonferenza bacino e fattori di rischio cardiovascolare in una popolazione del sud Italia.

AIM: To evaluate the relationship between body mass index (BMI), body fat distribution and some coronary heart disease risk factors like hyperlipidemia, hypertension and cigarette smoking. STUDY DESIGN: Cross-sectional. PLACE: Tiriolo, a little town close to Catanzaro, of prevalent rural economy. PARTICIPANTS: Volunteers, both males and females, aged more than 30 years and living in Tiriolo. MEASUREMENTS: Body weight and height with subjects in ordinary street clothes and without shoes. Systolic (SBP) and diastolic blood pressure (DBP) by a zero-random sphygmomanometer. Total (TC) and HDL cholesterol (HDL-C) using fingerstick capillary sample technology by a Cholestech analyzer. Waist circumference (W), measured midway between the lower rib margin and the iliac crest, and hip circumference (H) measured at the widest point over the greater trocanthers. Smoking habit by questionnaire. RESULTS: Females had higher values of SBP, DBP, BMI and HDL-C and lower of TC/HDL-C ratio and W/H ratio. Age was similar in both sexes. Females had lower prevalence of hyperlipidemia and cigarette smoking and higher prevalence of hypertension. BMI was strongly associated to blood pressure levels whereas W/H ratio was correlated to TC/HDL-C ratio. CONCLUSION: BMI and W/H ratio give complementary information, useful to assess the cardiovascular risk profile. The simplicity and quickness of these measurements should lead to their large utilization both in epidemiological prevention studies and everyday clinical practice.

High-level expression and in vitro mutagenesis of a fibrillogenic 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein.

We amplified DNA encoding the 3' 109 codons of Alzheimer's-disease amyloid precursor protein (APP) inclusive of the beta protein (A beta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13. We also modified this construct to generate recombinant molecules incorporating two recently described APP mutants by site-directed mutagenesis. Both native C109 (deletion construct inclusive of the C-terminal 109 residues of APP) and constructs with a single mutation at codon 642 (T-->G, resulting in a substitution of glycine for valine) or a double mutation at codons 595 (G-->T, substituting asparagine for lysine) and 596 (A-->C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20% of total bacterial protein after induction. The major constituent of expressed C109 protein had an apparent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the full-length construct by size and N-terminal microsequencing. Also present was a 4-5 kDa species that co-purified with C109, constituting only approximately 1% of expressed protein, which was revealed by Western-blot analysis with antibodies specific for A beta epitopes and after biotinylation of purified recombinant C109. This fragment shared N-terminal sequence with, and appeared to arise by proteolysis of, full-length C109 in biosynthetic labelling experiments. C109 spontaneously precipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5%) fibrillar component that was reactive with antibodies to a C-terminal epitope of APP. Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal A beta-inclusive fragments of APP that have been found in transfected cells, brain cortex and cerebral microvessels.

Cross-reactive antigenic domains of the flagellin protein of Borrelia burgdorferi.

The p41 flagellin of Borrelia burgdorferi is the most common antigen recognized by serum of patients with Lyme borreliosis. This antigen shares amino acid homology, particularly in the amino and carboxy termini, with periflagellar antigens found in other microorganisms including Treponema pallidum. We cloned and expressed the p41 open reading frame in Escherichia coli and expressed it both as TrpE fusion and full-length unfused proteins. Also, we generated deletion constructs of various portions of the gene. Sera from patients with late Lyme borreliosis and secondary syphilis were used to identify the recombinant proteins by immunoblot analysis. Sera from 26 patients with Lyme borreliosis, 20 with secondary syphilis and 10 controls were used to identify cross-reactive domains of the B. burgdorferi flagellin. The variable region (amino acids 131-234) of the protein was recognized by 59% (15/26) of patients with late Lyme borreliosis compared to 30% (6/20) of patients with secondary syphilis and no (0/10) control patients. It appears that cross-reactive epitopes between B. burgdorferi and T. pallidum extend to the variable region of the flagellin.

In situ localization of PCR-amplified human and viral cDNAs.

We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby RNA may be nonisotopically detected in fixed cells when amplified by PCR after cDNA synthesis by RT. RT in situ PCR using primers specific for the measles virus generated an intense signal in most measles-infected HeLa cells, as compared to the weak signal generated in few cells using standard in situ hybridization analysis. The viral RNA that localized to the nucleus spared the nucleoli, was most evident when the RT step used the primer complementary to the negative genomic strand, and was demonstrated in all multinucleated cells and the majority of uninucleate cells. A hybridization signal was evident with standard RNA in situ hybridization using the human megakaryocyte cell line Dami and a probe for glycoprotein IIB (GIIB) mRNA but not a probe for amyloid precursor protein (APP) or gelsolin (GEL) mRNA. After RT in situ PCR, signals were evident for each target localizing to the nucleolus for APP and to perinucleolar and cytoplasmic locations for GEL and GIIB. The latter findings suggest that mRNAs may follow different geographic pathways as they progress from premessage to transcriptionally active message.

Characterization of a Borrelia burgdorferi dnaJ homolog.

The gene encoding a Borrelia burgdorferi DnaJ homolog, located immediately 3' of the hsp70 gene, was characterized. Although there is a single copy of the dnaJ gene on the spirochetal chromosome, two distinct dnaJ transcripts are detected in B. burgdorferi RNA. RNA blot analysis indicates that the dnaJ gene can be transcribed alone or as part of a larger transcript containing the hsp70 homolog.