Detection and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies to HIV p24gag

A panel of monoclonal antibodies (Mabs) was used to analyze the number and localization of B-cell epitopes on human immunodeficiency virus (HIV) p24gag and the variability of these epitopes in sequential HIV isolates and in isolates from different geographical origin. The specificity of these Mabs was demonstrated by immunoblotting and radioimmunoprecipitation assays. Cross-inhibition experiments indicated the presence of at least five different epitopes on p24. Analysis with p24 recombinant products revealed that three of the Mabs to p24 were directed to epitopes localized on the C-terminal part. Four other Mabs were directed to epitopes localized on the N-terminal half of the protein. Anti-p24 Mabs were used to develop HIV p24 antigen-capture assays. Application of these assays in HIV isolation resulted in more efficient recovery of HIV. Serotyping of HIV-1 isolates with five anti-p24 Mabs demonstrated that 55/65 isolates recovered from Dutch and Belgian individuals, but only 4/9 HIV-1 African isolates, were recognized by all five Mabs. Five of nine Central African HIV-1 isolates were not reactive with at least one of these Mabs. The variability of p24 appeared to be predominantly localized on the N-terminal part of the protein. Lack of expression of antigenic determinants on p24 was shown to be independent of culture conditions. Moreover, an infectious molecular clone was shown to have the same serotype as the corresponding HIV isolate. The serotype of sequential isolates obtained from 17 individuals over a 1 1/2- to 2 1/2-year period did not change, suggesting a limited in vivo p24 variation over time.     

The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.

A characteristic of patients with autoimmune diseases such as Sj?gren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.     

Immunogenicity of 20 μg of recombinant DNA hepatitis B vaccine in healthy neonates: A comparison of three different vaccination schemes

The immunogenicity of a full dose (20 μg) of recombinant DNA yeast-derived hepatitis B vaccine (Engerix-B) was assessed in healthy neonates in order to compare three candidate vaccination schemes. After randomization 162 newborns of hepatitis B surface antigen (HBsAg) negative mothers entered the study. Neonates received hepatitis B vaccine according to a fourdose vaccination scheme starting either at month 3 (scheme I: months 3,4,5, and 11) or at birth (scheme III: months 0,1,2, and 11). Another group of neonates received hepatitis B vaccine according to a three-dose scheme starting at birth (scheme II: months 0, 1, and 6). Serious adverse reactions were not observed; 2.5% of the vaccinated newborns suffered mild transient local symptoms. The vaccine was highly immunogenic irrespective of vaccination scheme; all infants developed anti-HBs levels ≥10 IU/L, 97% ≥100 IU/L. The immunogenicity of hepatitis B vaccine after primary and booster vaccinations, administered in the four-dose scheme started at birth, was significantly higher (P< 0.05) than in the three-dose scheme started at birth. Hepatitis B vaccination according to the four-dose scheme started at month 3 produced significantly higher (P < 0.05) antibody levels in comparison to the four-dose scheme started directly after birth. This study showed that a fourdose hepatitis B vaccination scheme starting at month 3 resulted in the highest antibody levels of the three schemes investigated and can be recommended for incorporation in the Expanded Programme on Immunization in The Netherlands.     

Predictive value of neonatal MRI as compared to ultrasound in premature infants with mild periventricular white matter changes

A follow-up study was performed in 42 premature infants in whom serial neonatal ultrasound and a single neonatal MRI of the brain was normal, or showed mild periventricular white matter changes. The aim of the study was to evaluate the clinical significance of periventricular signal intensity changes on MRI and to compare the predictive value of neonatal MRI with that of ultrasound. The infants underwent repeated standardised motor assessments and developmental tests. MRI was repeated at the corrected age of 12 months. Pronounced periventricular signal intensity changes on neonatal MRI and periventricular echodensities (flaring) on ultrasound were associated with a high incidence of transient motor problems during infancy. The degree of echogenicity carried the highest predictive value, as compared to duration of flaring on ultrasound and degree of periventricular signal intensity change on MRI. It is concluded that signal intensity changes on neonatal MRI represent the same ischaemic change of the periventricular white matter as flaring on ultrasound and that routine neonatal MRI screening is not warranted in premature infants without clinical evidence of neurological problems and with normal or mildly abnormal ultrasound scans. Recording of the degree of echogenicity should become a routine procedure in neonatal cerebral ultrasonography.     

Malignant Cytological Washings from Radical Prostatectomy Specimens: A Possible Mechanism for Local Recurrence of Prostate Cancer Following Surgical Treatment of Organ Confined Disease

Purpose

Local recurrence of prostate cancer following complete and successful resection of organ confined disease has been variably reported in men. We hypothesized that observed secretions from the cut distal urethra during radical prostatectomy may contain malignant prostatic epithelial cells and contribute to this problem.

Materials and Methods

A prospective study was done of prostate cytology specimens from 50 consecutive men with clinically organ confined adenocarcinoma of the prostate undergoing radical retropubic or radical perineal prostatectomy. Direct cytological evaluation by 1 examiner was used to identify malignant or benign cells in these washings.

Results

Of 33 radical perineal and 17 radical retropubic prostatectomy specimens organ confinement was confirmed in 58 percent. Malignant prostatic epithelial cells were observed in 24 percent of all cytology specimens. Of cytological washings from prostates with pathologically confirmed organ confined cancers 17 percent showed malignant cells. While perineural invasion was noted in a majority of tumors with positive washings, only Gleason grade was a statistically significant predictor of recurrence (p = 0.009). Surgical approach did not alter the rate of positive cytology.

Conclusions

Malignant prostatic epithelial cells can be identified in the prostatic washings from men with pathologically organ confined prostate cancer. Surgical approach did not change the cytological findings. Gleason grade is a statistically significant predictor of cytological malignancy. These cells may represent a mechanism of failure following successful radical prostatectomy.     

Formulation of a stable vidarabine infusion fluid

Decomposition of vidarabine in a 5% glucose infusion fluid after steam sterilization was measured byhplc andtlc analysis. After sterilization for 60 minutes at 100°C and 20 minutes at 120°C no degradation could be observed. A slight but significant degradation was observed after sterilization for 60 minutes at 120°C followed by storage for eight months. Assuming 5% loss of content to be acceptable, it was concluded that a vidarabine infusion fluid can be sterilized for 20 minutes at 120°C and stored at room temperature for at least eight months afterwards.     

Abstracts from the XIIth Scientific Meeting of the Argentine Society of Arterial Hypertension

Epidemiologic Survillance of Cardiovascular Disease Mollar G, Cavalieri L, Galarza C, Waisman G, Beratarrechea A, Petrlik E, Langlois E, Soriano F, Marchetti M, and Gonzalez B de Quirós F Programas Médicos de Plan de Salud del Hospital Italiano de Buenos Aires

To enhance efficacy of health care, surveillance activities are required, especially with chronic prevalent diseases. Epidemiologic surveillance allows US to quantify and qualify health problems, settle priorities, identify high risk groups, manage and monitor health care systems, detect frequency changes in events, and assess performance of prevention and disease management programs. We have developed a surveillance system that identifies and reports daily on patients having cardiovascular disease (CVD) who are uncontrolled.

Objective: To evaluate the accomplishment of blood pressure (BP) measurement in people with CVD belonging to a health care system included in an epidemiological surveillance list.

Design: Cohort study between 1/1/2004 and 9/1/2004.

Methods: Patients 60 years old or older identified by surveillance system during the first term of 2004, were followed up. Patients were identified as having CVD through electronic medical record using international classification for primary attention (ICPA).

Patients are reported by the software system when presenting any of the following criteria: no blood pressure record during the past 6 months, blood pressure above 140/90 in the last record, and if diabetic A1C above 7.5%.

The connection with the appointment system allowed us to detect previously the patient’s attendance and, 15 min before his or her medical appointment, send the patient to be examined by the executors of a chronic disease program, according to the JNC VII guidelines.

The BP measurement is a common intervention to most chronic diseases because it increases cardiovascular risk, hence it is considered a process indicator of the surveillance system.

Results: Of the 24,411 patients having CVD, 5506 (26%) were listed during the first term, mean age was 74 years and 65% were female. By the end of 2004, 4660 (85%) patients had blood pressure recorded with an average values of 136.76 mmHg for systolic and 77.51 for diastolic blood pressure. Blood pressure was assessed three times in average.

Conclusion: Surveillance system allowed identifying of and intervening in a high proportion of patients.