New perspectives of quercetin and vitamin C effects on fibronectin-binding integrins and chemokine receptors in prostate cancer cell lines.

OBJECTIVES: The aim of this study is to investigate the effect of two abundant dietary supplements, quercetin and vitamin C on some factors involved in metastasis and proliferation of prostate cancer, which are resistant to conventional chemotherapies in late stages. BACKGROUND: Bone and brain are two common sites of metastases in prostate cancer, nevertheless the factors involved in their metastatic pathways are not well understood. METHODS: The effect of quercetin (75µM) and vitamin C (100 µM) on CXCR4, CXCR7 chemokine receptors, α4, α5 and ß1 integrins, ki-67 proliferation marker and Vascular endothelial growth factor, VEGF was evaluated using Quantitative Reverse Transcription PCR (RT-qPCR). RESULTS: The effect of quercetin and vitamin C alone was different on PC3 and DU145 prostate cancer cell lines, but sequential combination reduced significantly the expression of CXCR and CXCR7 chemokine receptors, α4, α5 and ß1 integrin subunits, VEGF and Ki-67 proliferation markers in PC3 and DU145 cell lines. CONCLUSION: Our results indicated the beneficial effect of quercetin and vitamin C on prostate cancer cells with different metastatic sites and their differential response to the treatment which in turn may lead us to reach suitable therapeutic outcomes to combat cancer (Fig. 3, Ref. 36).

Multiple model predictive control for optimal drug administration of mixed immunotherapy and chemotherapy of tumours.

BACKGROUND: Mixed immunotherapy and chemotherapy of tumours is one of the most efficient ways to improve cancer treatment strategies. However, it is important to 'design' an effective treatment programme which can optimize the ways of combining immunotherapy and chemotherapy to diminish their imminent side effects. Control engineering techniques could be used for this. METHODS: The method of multiple model predictive controller (MMPC) is applied to the modified Stepanova model to induce the best combination of drugs scheduling under a better health criteria profile. The proposed MMPC is a feedback scheme that can perform global optimization for both tumour volume and immune competent cell density by performing multiple constraints. RESULTS: Although current studies usually assume that immunotherapy has no side effect, this paper presents a new method of mixed drug administration by employing MMPC, which implements several constraints for chemotherapy and immunotherapy by considering both drug toxicity and autoimmune. With designed controller we need maximum 57% and 28% of full dosage of drugs for chemotherapy and immunotherapy in some instances, respectively. Therefore, through the proposed controller less dosage of drugs are needed, which contribute to suitable results with a perceptible reduction in medicine side effects. CONCLUSION: It is observed that in the presence of MMPC, the amount of required drugs is minimized, while the tumour volume is reduced. The efficiency of the presented method has been illustrated through simulations, as the system from an initial condition in the malignant region of the state space (macroscopic tumour volume) transfers into the benign region (microscopic tumour volume) in which the immune system can control tumour growth.

Reuse of spent FCC catalyst, waste serpentine and kiln rollers waste for synthesis of cordierite and cordierite-mullite ceramics.

Spent fluid catalytic cracking (FCC) was gathered from several petrochemical plants and calcined in a rotary furnace between 1000 and 1100°C in order to remove sulphur and hydrocarbon based impurities. Calcining process on FCC led to formation of AlVO4 ceramic phase, so converted the hazardous waste to non-hazardous applicable raw material. In this study, two ceramic bodies as cordierite and cordierite-mullite were synthesized with calcined spent FCC, waste serpentine, kiln rollers waste and high grade kaolin as raw materials. The XRD results showed that the cordierite and cordierite-mullite were synthesized successfully so that 96.4% of F1 (cordierite) sample fired at 1400°C was cordierite phase and F2 (cordierite-mullite) sample fired at 1450°C was completely cordierite and mullite phases. The synthesized cordierite and cordierite-mullite samples had lower porosity values and coefficient of thermal expansion (CTE) than similar industrial products. The negative CTE value that obtained from the cordierite sample up to 800°C is favorable for some applications. The considerable results of the synthesized cordierite and cordierite-mullite from this work present cost reduction of the two ceramic bodies production and may help to solve the environmental problems with the use of three waste sources in large scales.

Gut Microbiome in Chronic Kidney Disease.

With over 100 trillion microbial cells, the gut microbiome plays important roles in both the maintenance of health and the pathogenesis of disease. Gut microbiome dysbiosis, resulted from alteration of composition and function of the gut microbiome and disruption of gut barrier function, is commonly seen in patients with chronic kidney disease (CKD). The dysbiotic gut microbiome generates excessive amounts of uremic toxins, and the impaired intestinal barrier permits translocation of these toxins into the systemic circulation. Many of these uremic toxins have been implicated in the progression of CKD and increased cardiovascular risk. Various therapeutic interventions have been proposed that aim to restore gut microbiome symbiosis. If proven effective, these interventions will have a significant impact on the management of CKD patients. In this review, we discuss the consequences of gut microbiome dysbiosis in the context of CKD, discuss the consequences of gut dysbiosis, and highlight some of the recent interventions targeting the gut microbiome for therapeutic purposes.

Beneficial effects of omega-3 and vitamin E coadministration on gene expression of SIRT1 and PGC1α and serum antioxidant enzymes in patients with coronary artery disease.

BACKGROUND AND AIM: SIRT1 and PGC1α are two important genes, which play critical roles in regulating oxidative stress and inflammation processes. The study aimed assess the effects of coadministration of omega-3 and vitamin E supplements on SIRT1 and PGC1α gene expression and serum levels of antioxidant enzymes in coronary artery disease (CAD) patients. METHODS AND RESULTS: Participants of this randomized controlled trial included 60 CAD male patients who were categorized into three groups: Group 1 received omega-3 (4 g/day) and vitamin E placebo (OP), group 2 omega-3 (4 g/day) and vitamin E (400 IU/day; OE), and group 3 omega-3 and vitamin E placebos (PP) for 2 months. Gene expression of SIRT1 and PGC1α in peripheral blood mononuclear cells (PBMCS) was assessed by reverse transcription polymerase chain reaction (RT-PCR). Furthermore, serum antioxidant enzyme and high-sensitivity C-reactive protein (hsCRP) levels were assessed at the beginning and end of the intervention. Gene expression of SIRT1 and PGC1α increased significantly in the OE group (P = 0.039 and P = 0.050, respectively). Catalase and hsCRP levels increased significantly in the OE and OP groups. However, glutathione peroxidase (GPX) and superoxide dismutase (SOD) levels did not statistically change in all groups. The total antioxidant capacity (TAC) increased significantly in the OE group (P = 0.009) but not in OP and PP groups. CONCLUSION: Supplementation of omega-3 fatty acids in combination with vitamin E may have beneficial effects on CAD patients by increasing gene expression of SIRT1 and PGC1α and improving oxidative stress and inflammation in these patients.

Comparison of desmoglein 1 and 3 enzyme-linked immunosorbent assay and direct immunofluorescence for evaluation of immunological remission in pemphigus vulgaris.

BACKGROUND: Drug withdrawal is the ultimate goal in the management of patients with pemphigus. Direct immunofluorescence (DIF) has long been considered the gold-standard test to predict immunological remission in pemphigus vulgaris (PV); however, there have been no comparisons between DIF and antidesmoglein (anti-Dsg) ELISA. AIM: To compare anti-Dsg ELISA with DIF in patients with PV for evaluation of immunological remission. METHODS: The study enrolled 46 patients with PV who had absence of any lesion, and had a daily prednisolone dosage of ≤ 10 mg without adjuvant drug treatment in the preceding 6 months. Biopsy specimens were taken from patients and processed for DIF. Intercellular deposition of IgG and/or C3 was considered positive. Serum samples were also collected for anti-Dsg1 and anti-Dsg3 ELISA, and an ELISA index value of > 20.0 was considered positive. RESULTS: DIF and anti-Dsg ELISA were positive for 11 (23.9%) and 18 patients (39.1%), respectively. Anti-Dsg ELISA had a sensitivity of 100%, a specificity of 80%, a positive predictive value of 61.1% and a negative predictive value of 100%. CONCLUSIONS: The high sensitivity of anti-Dsg ELISA proves that this simple serological test is a good substitute for DIF for evaluation of immunological remission in PV. As none of the DIF-positive patients was anti-Dsg-negative, it is possible that during the course of immunological remission, results for DIF may become negative before the results for Dsg ELISA do so.

A study of leptin serum concentrations in patients with major Beta-thalassemia.

BACKGROUND: The aim of this study was to evaluate leptin serum levels in patients with major beta thalassemia which was also associated with their ferritin serum levels. MATERIALS AND METHODS: This case-control study was performed on 90 children -6 months to 16 years old, in Zabol, Amir- al- Momenin Hospital. Patients were divided in two groups and were matched in age and sex. All Children were examined and those eligible children who had not known heart disease, iron deficiency anemia, kidney disease, diabetes, fever and systemic diseases were enrolled after taking the informed consent of their parents. After collecting the samples, leptin and ferritin levels of the serum were measured in two groups by ELISA method. Then, the data was analyzed by the related statistical tests and SPSS 20 software. RESULTS: The mean of the serum levels of leptin and ferritin showed a significant difference in the case and control groups (P-value<0.05). An inverse statistical correlation was found for the serum levels of leptin and ferritin among the studied groups (P-value<0.05). Levels of leptin in the case group showed a significant gender difference (P-value<0.05), while based on BMI and age, no significant difference was observed for the serum levels of leptin in the case group. CONCLUSION: Based on the results of this study, major thalassemia reduces serum levels of leptin regardless of age and body mass. The study also found an inverse statistical correlation between serum levels of leptin and ferritin among the studied people.

A comparative study of conjunctival autograft and minimally invasive pterygium surgery in primary pterygia.

This study aimed at comparing the results of the Conjunctival Autograft Technique (CAG) and Minimally Invasive Pterygium Surgery (MIPS) in primary pterygium excisions. This was a prospective randomized clinical study performed during a one-year period (2009-2010). One hundred and twenty two patients with primary pterygium were randomized in 2 groups: group A (CAG) including 36 patients and group B (MIPS) including 86 patients. The two groups were compared considering the recurrence rate and probable complications of the procedures. Recurrences were detected in 4 patients (11.1%) in group A and 5 patients (5.8%) in group B with no significant difference in this regard (p = 0.447). No major complications occurred during the follow-up period. This study showed that acceptable recurrence- free rates could be achieved (albeit nonsignificant) using MIPS technique in patients with primary pterygium and can be considered as a good alternative in the surgical management of pterygia because of its simplicity and low surgical time.

Genetic diversity of grapevine accessions from Iran, Russia and USA using microsatellite markers.

To discover marker information content and differentiation among grapevine accessions from Iran, USA and Russia, nine microsatellite markers were used. A total of 75 alleles were detected, giving a mean of 8.3 alleles per 9 loci. The total number of alleles per locus varied between 6 to 11 and the polymorphism information content ranged from 0.65 to 0.88, indicating that these loci were highly informative. A positive correlation (r = 0.870) was observed between the number of alleles and the level of polymorphism. Two SSRs loci including SSrVrZAG47 and VVMD27 were found to be probably synonymous. Gene diversities were high in all populations with values ranging from 0.709 to 0.784. In all populations, the mean number (averaged over loci) of heterozygous individuals was higher than expected. PCO analysis could not be so clearly differentiated accessions from Iran and Russia. The pattern of clustering of the Vitis vinifera populations was according to their geographic distribution. It is suggested that accessions could possibly be assigned to their regions of origin according to their genotypes.

Frequency of isolated hepatitis B core antibody in HIV-hepatitis C virus co-infected individuals.

Association between isolated hepatitis B core antibody (anti-HBc) and hepatitis C virus (HCV) infection has been noted in HIV-infected individuals. This study describes the frequency of isolated anti-HBc and its possible value for the detection of HBV-DNA in HIV-infected patients with or without HCV co-infection. Ninety-two HIV-infected patients were enrolled in the study. Hepatitis B surface antigen (HBs Ag), anti-HBs, anti-HBc, anti-HCV, HIV viral load and CD4 count were tested in all subjects. Then we compared 63 subjects with HIV-HCV co-infection with 29 subjects with HIV infection alone regarding isolated anti-HBc (HBs Ag negative, anti-HBs negative and anti-HBc positive). The presence of HBV-DNA was determined by real-time polymerase chain reaction in serum samples of patients with isolated anti-HBc. Of 63 anti-HCV-positive patients, 18 subjects (28.6%, 95% [confidence interval] CI: 22.6-34.6%), and of 29 anti-HCV-negative patients, five subjects (17.2%, 95% CI: 11.5-22.9%) had isolated anti-HBc. HBV-DNA was detectable in three of 18 anti-HCV-positive patients (16.7%, 95% CI: 9.7-23.7%) and none of the anti-HCV-negative patients with isolated anti-HBc. Our study showed that individuals co-infected with HIV and HCV were more likely to have isolated anti-HBc than subjects with HIV alone. This investigation also demonstrates that the presence of isolated anti-HBc in HIV-HCV-infected individuals may reflect occult HBV infection in these patients.

Efficacy and long-term immunogenicity of hepatitis B vaccine in haemodialysis patients.

BACKGROUND: Hepatitis B vaccine is effective in protection against hepatitis B virus (HBV) infection in haemodialysis (HD) patients, but the antibody response is variable in this population and the persistence of immunity in them remains largely unknown. In this study we aimed to evaluate the efficacy and long-term immunogenicity of hepatitis B vaccine in HD patients. METHODS: In this study, we initially offered HBV vaccination as double dose, four vaccine series schedule (40 microg injections intramuscularly in the deltoid muscle at 0, 1, 2 and 6 months) to 54 HD patients who were negative for hepatitis B core antibody and did not receive any dose of HBV vaccine previously. Serum levels of hepatitis B surface antibody (anti-HBs) tested 1-2 months after completion of vaccination. Then we follow the patients up to 1 year after primary vaccination to evaluate the persistence of immunity (as indicated by serum levels of anti-HBs higher than or equal to 10 IU/l). RESULTS: After primary vaccination, 87% of patients developed anti-HBs levels above 10 IU/l. 27.8% and 59.2% of them were weak responders and high responders respectively. 13% of patients were non-responders. After 1-year follow-up, 18.18% of responders had lost their anti-HBs (transient responders). All of them were initially in weak responders group and had lower anti-HBs levels. CONCLUSION: We found an average percentage of seroconversion after primary HBV vaccination in HD patients. Our study also supported this fact that an antibody titre above 100 IU/l following primary vaccination is necessary to maintain that level of antibody 1 year later.

Abortive aphthous-like oral lesions: an underreported initial presentation of pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) usually presents as non-healing, painful oral erosions, but transient or aphtha-like lesions are not exceptional at the very beginning of the disease leading to the common misdiagnosis of recurrent aphthous stomatitis (RAS). We designed this cross-sectional, questionnaire-based study to evaluate this underreported yet important presentation of PV. MATERIAL AND METHODS: One hundred and eighty-five consecutive PV patients were interviewed and a questionnaire, comprising items related to the natural history of oral lesions, was filled in for each. Fourteen patients who had taken steroids (topical or systemic) before their final diagnosis were excluded. RESULTS: Twenty-three per cent of patients gave a history of transient aphthous-like lesions; 95% of them were misdiagnosed as aphthae. These lesions were especially reported by patients aged 40 years or older (P < 0.047). CONCLUSION: PV should be kept in mind as a rare differential diagnosis of transient oral ulcerations. We recommend careful observation of these patients and performing indirect immunofluorescence or desmoglein ELISA and even biopsy in atypical cases, to rule out PV especially in older patients and predisposed ethic groups. To find out the differential aspects of RAS and aphthous-like PV, a cohort study on RAS patients is suggested.

Posterior sub-tenon triamcinolone for refractory diabetic macular edema: a randomized clinical trial.

PURPOSE: To evaluate the effect of posterior sub-tenon triamcinolone acetonide (TA) injection on clinical, angiographic, and optical coherence tomographic (OCT) parameters in refractory diabetic macular edema (DME). METHODS: In a double-masked placebo-controlled clinical trial, 64 eyes were randomly assigned to two groups. The treatment group (32 eyes) received 40 mg posterior sub-tenon injection of TA and the placebo group (32 eyes) received subconjunctival injection of a placebo. The injections were repeated after 2 months in both groups. Complete ophthalmologic examination, fluorescein angiography, and OCT were performed before intervention and after 4 months. Quantitative measurement of angiographic variables such as the amount of hard exudates (HE), size of foveal avascular zone (FAZ), and leakage severity was performed by computer, using Photoshop software. RESULTS: Initial best-corrected visual acuity (VA) was 0.93+/-0.39 logMAR in the placebo group and 0.75+/-0.38 logMAR in the treatment group. At 4 months, corrected VA was 0.88+/-0.48 logMAR in the controls versus 0.71+/-0.42 logMAR in the cases. Mean central macular thickness measured by OCT before and 4 months after injection was 392 and 377 microns in the treatment group and 388 and 357 microns in the placebo group, respectively. No statistically significant difference was detected between the two groups. The difference was also not significant in HE, FAZ, and leakage in the angiograms. CONCLUSIONS: Two injections of posterior sub-tenon TA had no therapeutic effect on refractory DME.

Effect of tranexamic acid on early postvitrectomy diabetic haemorrhage; a randomised clinical trial.

AIMS: To evaluate the effect of tranexamic acid on early postvitrectomy haemorrhage in diabetic patients. METHODS: In a clinical trial, 62 diabetic patients scheduled for vitrectomy were randomly assigned to two groups. The treatment group (32 eyes) received two doses of tranexamic acid (10 mg/kg) shortly before and after the operation intravenously, continued orally for 4 days (20 mg/kg/8 hours). The control group (30 eyes) received no medication. Both media clarity and visual acuity were compared during 4 weeks. RESULTS: Four weeks after surgery visual acuity was low (< or =1 metre counting fingers) in 21.4%, moderate (>1 metre counting fingers but<20/200) in 14.3%, and good (> or =20/200) in 64.3% of the treated group. Corresponding figures in the control group were 26.1%, 26.1%, and 47.8%, respectively. These differences were of no statistical significance. The ratio of mild to severe vitreous haemorrhage during the first 4 days and after 4 weeks was 79% to 21% and 82% to 18% in the treatment group and 76.7% to 23.3% and 78.3% to 21.7% in the control group respectively, which showed no statistically significant difference. CONCLUSION: Tranexamic acid, with the method of administration in this study, had no effect on reducing early postvitrectomy haemorrhage in diabetic patients.

Rhesus monkey placental transgene expression after lentiviral gene transfer into preimplantation embryos.

Transgenic mice have provided invaluable information about gene function and regulation. However, because of marked differences between rodents and primates, some areas of human biology such as early embryonic development, aging, and maternal-fetal interactions would be best studied in a nonhuman primate model. Here, we report that gene transfer into rhesus monkey (Macaca mulatta) preimplantation embryos gives rise to transgenic placentas that express a reporter transgene (eGFP). Blastocysts resulting from culture of in vitro fertilized ova were transduced with a self-inactivating lentiviral vector and transferred into recipient females. One twin and one singleton pregnancy were produced from a single stimulation cycle, and one live rhesus monkey was born from each pregnancy. Placentas from all conceptuses showed expression of the transgene as detected by reverse transcription-PCR, ribonuclease protection assay, direct epifluorescence, immunohistochemistry, and Western blot analysis. Integration in somatic tissues of the offspring was not detected. A maternal immune response to the xenogeneic placental antigen was shown by the presence of anti-GFP antibodies in peripheral blood of the recipient females by day 99 of gestation (term = 165 days). These results demonstrate that transgene expression during gestation is compatible with successful pregnancy in nonhuman primates and provides an approach that could be broadly applicable to the development of novel models for primate biomedical research.

Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins.

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.

Lentiviral vectors for enhanced gene expression in human hematopoietic cells.

Accumulated data indicate that current generation lentiviral vectors, which generally utilize an internal human cytomegalovirus (CMV) immediate early region enhancer-promoter to transcribe the gene of interest, are not yet optimized for efficient expression in human hematopoietic stem/progenitor cells (HSPCs). As a first step toward this goal, we constructed self-inactivating derivatives of the HIV-1-based transfer vector pHR' containing the enhanced green fluorescent protein (GFP) gene as reporter and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). GFP expression was driven by a variety of strong viral and cellular promoters, including the murine stem cell virus (MSCV) long terminal repeat (LTR), a Gibbon ape leukemia virus (GALV) LTR, the human elongation factor 1alpha (EF1alpha) promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), and the human phosphoglycerate kinase 1 (PGK) promoter. In contrast to results obtained in human embryonic kidney 293T cells and fibrosarcoma HT1080 cells, in which the CMV promoter expressed GFP at the highest levels, significantly higher levels of GFP expression (3- to 5-fold) were achieved with the MSCV LTR, the EF1alpha promoter, and the CAG promoter in the human HSPC line KG1a. Removal of the WPRE indicated that it stimulated GFP expression from all of the vectors in KG1a cells (up to 3-fold), although it only marginally improved the performance of the intron-containing EF1alpha and CAG promoters (<1.5-fold stimulation). The vectors using the MSCV LTR, the GALV LTR, and the PGK promoter were the most efficient at transducing primary human CD34+ cord blood progenitors under the conditions employed. High-level GFP expression in the NOD/SCID xenograft model was demonstrated with the pHR' derivative bearing the MSCV LTR. These new lentiviral vector backbones provide a basis for the rational design of improved delivery vehicles for human HSPC gene transfer applications.

Targeted RNases: a feasibility study for use in HIV gene therapy.

A targeted RNase would be ideal for gene therapy of several acquired and inherited disorders. Such an RNase may be engineered to contain a ribonucleolytic domain and a specific target RNA binding domain. To demonstrate the feasibility of this approach, an RNase targeted against human immunodeficiency virus (HIV) RNA--Tev-RNase T1--was designed and tested for its use in HIV-1 gene therapy. A human CD4+ T lymphoid (MT4) cell line and human peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors lacking or expressing the tevT1 gene. Expression of enzymatically functional Tev-RNase T1 protein and its lack of toxicity was demonstrated in stable MT4 transductants. Compared with control cells lacking this protein, both transduced MT4 cells and PBLs expressing Tev-RNase T1 delayed HIV-1 replication. Tev-RNase T1 was shown to act after integration, since HIV-1 proviral DNA could be detected, but the amount of HIV-1 RNA produced in MT4 cells and PBLs was significantly decreased. This study demonstrates the feasibility of a targeted RNase strategy for therapeutic use.

Inhibition of HIV-1 replication by retroviral vectors expressing monomeric and multimeric hammerhead ribozymes.

Retroviral vectors were engineered to express monomeric and multimeric hammerhead ribozymes targeting one and nine highly conserved sites within the HIV-1 envelope (Env) coding region. In vitro, both the monomeric and multimeric ribozymes were shown to be active and cleave the target RNA containing the cleavage sites. A human CD4+ T lymphocyte-derived MT4 cell line was stably transduced with retroviral vectors expressing these ribozymes. Ribozyme expression in stably transduced cells was confirmed by Northern blot analysis and reverse-transcription polymerase chain reaction (RT-PCR). As compared with the control cells lacking any ribozyme, HIV-1 replication was delayed in monomeric RzEnv-expressing cells. Virus replication was almost completely inhibited in multimeric RzEnv1-9-expressing cells as no viral RNA or protein could be detected in these cells and in their culture supernatants for up to 60 days after infection. The genomic DNA from RzEnv1-9-expressing cells was shown to contain HIV-1 proviral DNA sequences at days 3 and 60 after HIV infection. HIV-1 used in the challenge experiments was found to contain fully reverse transcribed '-' strand DNA which should have been able to infect complete proviral DNA synthesis and integrate within the cellular genome without being affected by pre-existing ribozymes. Therefore, the proviral DNA present at day 3 after infection may have originated from infection by such DNA-containing virus particles. The results obtained with the retroviral vector expressing RzEnv1-9 are very encouraging and we envisage its future use in anti-HIV-1 gene therapy.