Preclinical ex vivo expansion of peripheral blood CD34+ selected cells from cancer patients mobilized with combination chemotherapy and granulocyte colony-stimulating factor.

BACKGROUND AND OBJECTIVES: Ex vivo peripheral blood progenitor cell (PBPC) expansion has been proposed as a strategy to increase the number of haematopoietic progenitors available for cell transplantation. We have expanded CD34+ cells from PBPCs obtained from four patients with haematological malignancies and one patient with an Ewing's sarcoma. MATERIALS AND METHODS: Cells were expanded in the Dideco 'Pluricell system'. After 12 days in culture, we evaluated cell phenotype, total nucleated cells, CD34+ fold increase, cell apoptosis and colony assay of expanded cells. Cell engraftment has been evaluated by transplanting two groups of irradiated non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice with expanded and non-expanded cell populations. RESULTS: Total nucleated cells and CD34+ cells increased 59.5 and 4.0 times, respectively. The expanded cells were mainly constituted of myeloid and megakaryocytic cells. A significant increase in the number of colony-forming unit-granulocyte macrophage (CFU-GM) was observed in the CFU assay. Ten mice transplanted with expanded cells showed a best overall survival (80%) compared to 10 mice transplanted with non-expanded cells (20%). Human CD45+ cells were detected by flow cytometry and polymerase chain reaction in bone marrow and spleen of transplanted animals. The relative low engraftment level obtained with the expanded cells suggests a loss of SCID repopulating cells maybe due to cell differentiation during expansion. CONCLUSIONS: We have demonstrated the feasibility of the ex vivo expansion of mobilized PBPCs from cancer patients, evidencing a clonal expansion of CFUs and the ability of the expanded cells to engraft the bone marrow and spleen of immunosuppressed mice. The differentiation of the CD34+ stem cell compartment could be further minimized by ameliorating the expansion conditions.

Ex vivo expansion of umbilical cord blood CD34 cells in a closed system: a multicentric study.

BACKGROUND AND OBJECTIVES: The Dideco 'Pluricell System' is a CE-marked medical device allowing haematopoietic stem cell (HSC) expansion. It comprises a kit of cGMP cytokines and reagents, a closed-cell expansion chamber and a cell-washing set. We tested the system in a multicentric study by expanding CD34(+) cells from eight fresh umbilical cord blood (UCB) samples. MATERIALS AND METHODS: During culture, the mean nucleated cell (NC) count, the mean CD34(+) cell count, fold expansion, viability and apoptosis were measured. Clonogenic assays and immunophenotypical characterization were performed on days 0, 7 and 12. On the expanded cellular product, in three cases cell genotyping, endotoxin level and mycoplasma detection (by polymerase chain reaction) were performed. RESULTS: The mean CD34(+) cell expansion on days 7 and 12 was sevenfold and 12-fold respectively and the mean NC expansion was 69-fold and 180-fold. The mean NC viability on day 12 was 96.9% (94.4-99.1). After 12 days, granulocyte-macrophage colony-forming units (GM-CFU) showed a 20-fold increase: a slight increase in CD34(+) cell apoptosis was observed during culture. In all of three cases neither chromosomal alterations nor mycoplasma contamination was detected. No significant endotoxin levels were detected after expansion. CONCLUSIONS: The device allows the ex vivo expansion of NC and CD34(+) cells in a closed system. The expanded cellular product is a mixture of progenitors (CD34(+) cells) and differentiated (mainly myeloid and megakaryocytic) cells. To reduce cell apoptosis, more frequent cell feeding during culture should be tested.

Evaluation of ex vivo expansion and engraftment in NOD-SCID mice of umbilical cord blood CD34+ cells using the DIDECO "Pluricell System".

The Dideco "Pluricell System" is a commercially available closed device composed of an expansion chamber and a kit of certified reagents that allow haematopoietic stem cell expansion. We have expanded seven umbilical cord blood (UCB) samples following the manufacturer's instructions; two groups of irradiated NOD-SCID mice have been transplanted with expanded and nonexpanded cells from the same UCB, and bone marrow was analysed for the presence of human cells. Average UCB volume was 61.6+/-8.8 ml; mean nucleated cell content was 1090.5+/-189.9 x 10(6). Percentage and number of CD34+ cells were 0.37+/-0.13% and 3.9+/-1.2 x 10(6). After separation, CD34+ cell purity was 82+/-11%. Mean number of inoculated cells was 760 000; mean NC and CD34+ fold expansion at 12 days was 230.4+/-91.5 and 21.0+/-11.9. Both groups of mice showed successful engraftment: the percentage of human cells was higher in the group receiving expanded cells (3.4+/-2.01%) compared to the group receiving nonexpanded cells (1.5+/-0.66%) (P<0.00018, Mann-Whitney test). The cell population obtained after 12 days expansion consisted mainly of myeloid and megakaryocytic progenitors. The CD34+ antigen reached the maximum expression level at day 12 (7.5+/-2.0%). Analysis of lineage-markers for human myelomonocytic, megakaryocytic, B, T, CD34 and erythroid cells, gave evidence that all the lineages were represented in the marrow of transplanted mice.

Qualitative multiplex RT-PCR for simultaneous detection of hepatitis C virus and human immunodeficiency virus in plasma samples.

This report describes the development of a one-tube multiplex reverse transcriptase (RT)-PCR assay for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) in plasma samples. The assay was evaluated with two panels of HCV- and HIV-1-positive samples, as well as negative plasma specimens. Extraction and amplification of HCV and HIV-1 RNA from plasma samples were performed in a single reaction, and amplified genomes were detected with specific probes. Serial dilutions of the HCV and HIV-1 first World Health Organization International Standards were used to evaluate the sensitivity of the method. Two RNA controls were constructed to determine inter-assay variations and the sensitivity of the amplification step. The assay had good specificity and detected all the genotypes and subtypes tested. The analytical sensitivity of the entire assay was 100 IU/mL for HCV and 200 IU/mL for HIV-1, while the amplification step detected ten copies/reaction for HCV and 20 copies/reaction for HIV-1. The multiplex assay allowed the simultaneous extraction, amplification and detection of two virus genomes, thereby providing an important practical advantage and an efficient approach for analysing individual and pooled plasma donations.

Detection of human papillomavirus DNA and p53 gene mutations in esophageal cancer samples and adjacent normal mucosa.

BACKGROUND/AIM: There is evidence of a possible etiological role of human papillomaviruses (HPVs) in the development of esophageal tumors. Loss of function of the wild-type p53 tumor suppressor gene product by binding to E6 oncoproteins of high-risk HPVs is considered an important event in tumor development. The aim of this study was to verify the prevalence of HPV infection and p53 mutation in esophageal tumor tissue samples and in the adjacent normal mucosa in patients from a high-risk area in Italy. METHODS: DNA from 33 biopsy specimens (17 tumor sample biopsies and 16 samples of adjacent normal mucosa) was screened for HPV DNA using two polymerase chain reaction based procedures. Restriction fragment length polymorphism analysis was used for typing. Screening of p53 mutations was performed with polymerase chain reaction-single strand conformation polymorphism analysis and DNA sequencing. RESULTS: Overall, 8 of 17 patients presented HPV DNA; HPV 16 was detected in 4 of 8 samples. Samples from tumors and adjacent mucosa were positive for mucosal HPVs in 7 of 17 and 4 of 16 cases, respectively. In 1 case, HPV DNA was detected in the normal mucosa only. None of the samples contained HPVs of the epidermodysplasia verruciformis or cutaneous groups. Mutations of p53 were detected in two HPV DNA negative samples. In both cases, the mutation was present in the tumor only. CONCLUSIONS: Our results are in favor of the involvement of both aberrant p53 expression and HPV infection in the development of esophageal tumors. The high HPV infection rate in patients from a high-risk region suggests that subjects harboring HPVs (in particular HPV 16) in the esophagus should be considered at risk of esophageal malignancies.

A novel protocol that allows short-term stem cell expansion of both committed and pluripotent hematopoietic progenitor cells suitable for clinical use.

To obtain long-term engraftment and hematopoiesis in myeloablated patients, the cell population used for hematopoietic reconstitution should include a sufficient number of early pluripotent hematopoietic stem cells (HSCs), along with committed cells from the various lineages. For this purpose, the small subset of CD34+ cells purified from different sources must be expanded ex vivo. Since cytokines may induce both proliferation and differentiation, expansion would provide a cell population comprising committed as well as uncommitted cells. Optimization of HSC expansion methods could be obtained by a combination of cytokines able to sustain renewal of pluripotent cells yet endowed with poor differentiation potential. We used variations of the combinations of cytokines described by Brugger et al. [W. Brugger, S. Heimfels, R. J. Berenson, R. Mertelsmann, and L. Kanz (1995) N. Engl. J. Med. 333, 283-287] and Piacibello et al. [W. Piacibello, F. Sanavio, L. Garetto, A. Severino, D. Bergandi, J. Ferrario, F. Fagioli, M. Berger, and M. Aglietta (1997) Blood 89, 2644-2653] to expand UCB CD34+ cells and monitored proliferation rate and phenotype after 14 days of culture. Several hematopoietic lineage-associated surface antigens were evaluated. Our data show that flt3L and thrombopoietin in combination with IL-3, while sustaining a high CD34+ proliferation rate, provide a relatively low enrichment in very early uncommitted CD34+/CD38- cells. Conversely, in the absence of IL-3, they are less effective in inducing proliferation yet significantly increase the number of CD34+/CD38- cells. A combination of the above protocols, applied simultaneously to aliquots of the same sample, would allow expansion of both committed and pluripotent HSC. This strategy may represent a significant improvement for clinical applications.

Development and evaluation of a PCR-microplate capture hybridization method for direct detection of verotoxigenic Escherichia coli strains in artificially contaminated food samples.

For the purpose of detecting, directly in food, verotoxigenic Escherichia coli, a microplate hybridization method for the detection of PCR products from the SLT I and SLT II genes, was developed and evaluated. Two pairs of primers and two probes, specific for the SLT I gene and for the SLT II gene, were designed and tested. For the strains containing both genes, two PCR products of different molecular weights were obtained, whereas when only one gene was present only one fragment resulted from PCR. The use of the biotin-labeled probes allowed the immobilization of the PCR products in the microtiter plate wells and by this means their detection was possible using an ELISA-based technique. Forty artificially contaminated and fifty naturally contaminated food samples were analyzed by using the PCR-microplate hybridization technique developed in this study. All the artificially contaminated food samples were positive, independently of the number of cells inoculated before the enrichment step, whereas the naturally contaminated food samples were all negative.

PCR-RFLP-detected human papilloma virus infection in a group of senegalese women attending an STD clinic and identification of a new HPV-68 subtype.

Cancer of the cervix is the most common malignant tumor among women in Africa and, in particular, Senegal. Studies of the prevalence of human papilloma virus (HPV) infection in Africa have mainly focused on carcinomas. Data on the presence of the virus in women with normal cervical cytology are scarce. In this study, 158 cytologically normal women who had been referred to the 'Institut Pasteur de Dakar' (Senegal) for various genital complaints were investigated for the presence of HPV on exfoliated cells by PCR-RFLP. HPV was detected in 13.9% of cases. Oncogenic type HPV 16 was the most common type (40.9%), followed by HPV 53 and HPV 58, both detected in 13.6% of cases. Mixed HPV infections were present in 13. 6% of the subjects. Only HPVs belonging to the intermediate-high risk group were detected. These data suggest the need for careful cytological control of patients. A PCR-HPV fragment (GA115) possessing an original RFLP pattern was isolated. After sequencing, it showed a nucleotide homology of 97.1% with HPV 68 and should therefore be considered a new HPV 68 subtype. The use of PCR-RFLP strategy enables detection and typing of all known and as yet unknown genital HPVs. Variant and subtype classification of HPV types identified by oligonucleotide probe methods may need to be refined, especially for less prevalent HPVs and in areas where little information on HPV prevalence is available. More studies are needed to characterize satisfactorily the epidemiology of HPV in Africa.

A highly sensitive and fast non-radioactive method for the detection of polymerase chain reaction products from Salmonella serovars, such as Salmonella typhi, in blood specimens.

A polymerase chain reaction based test was developed for the detection of Salmonella spp. in blood specimens. After amplification of a 389 bp-polymerase chain reaction product from the invA gene, a microtiter plate hybridization assay was performed. The protocol described allowed the detection of six to seven copies of the Salmonella typhi genome, as determined by serial dilutions of DNA from S. typhi. Eighteen blood specimens from artificially infected rats and 22 blood specimens from patients were analyzed to validate the method. Considering that the most frequent Salmonella serovar isolated from blood in case of bacteremia is S. typhi, the polymerase chain reaction-microtiter plate hybridization technique could be used as a novel, rapid diagnostic method for typhoid fever, particularly when standard culture assays are negative.

Development of a PCR microplate-capture hybridization method for simple, fast and sensitive detection of Salmonella serovars in food.

The authors have developed an easy and rapid detection and identification system for Salmonella spp. in food. The gene inv A was selected as the target sequence. Oligonucleotides derived from conserved regions of this gene were able to exclusively prime the amplification of a 389 bp fragment when Salmonella spp. DNA was used as the template. An internal Salmonella spp. specific DNA probe was used for confirmation of the amplified polymerase chain reaction(PCR)product, by Southern blot or microplate-capture hybridization assay. In this fashion the sensitivity of the method was increased 100-fold (4.5 fg total DNA). To validate the method, a total of 75 food samples were tested. The PCR-microplate capture hybridization assay is easy to perform and much faster than traditional detection methods for Salmonella spp. in food. Hybridization in microtitre plates is more readily observed than in Southern blot and is more sensitive than conventional agarose gel electrophoresis.

Human papillomaviruses are commonly found in normal skin of immunocompetent hosts.

We have previously demonstrated, by the combined application of two degenerate polymerase chain reaction primer sets, the presence of human papillomavirus (HPV) DNA in 91% of cutaneous squamous cell cancers from renal allograft recipients, with multiple types being present in one-third of these tumors. Five HPV types--HPV 20, HPV 23, HPV 38, DL40, and DL267--accounted for 73% of positive results. These HPV types are all related to the epidermodysplasia verruciformis group, and HPV 38 was originally isolated from a melanoma. The aims of this study were to determine: (i) whether HPV DNA could readily be demonstrated in skin tumors, as well as in perilesional skin, of immunocompetent patients using two polymerase chain reaction primer sets; (ii) the prevalence of infections in normal skin; and (iii) the prevalence of HPV 38 or HPV 38 related viruses in melanoma. The HPV types detected in lesions from renal allograft recipient were present not only in the perilesional skin and tumors of immunocompetent patients, but also in 35% of normal skin biopsies. HPV DNA was present in 13% of the melanoma samples, but none harbored HPV 38 DNA. We identified four putatively new HPV types. Infections with different types of human papillomavirus are widespread and often occur in clinically normal skin. In vitro studies are required to determine the specific molecular mechanisms by which these HPV types may be involved in the etiology of nonmelanoma skin cancer.

Characterization of a putative new HPV genomic sequence from a cervical lesion using L1 consensus primers and restriction fragment length polymorphism.

Various methods have been proposed for HPV detection and typing. Prevalence and distribution among types have varied depending upon the methods used and the populations studied. We have applied the polymerase chain reaction (PCR) followed by a Restriction Fragment Length Polymorphism (RFLP) analysis using the MY09/MY11 primers for detection of HPV in cervicovaginal lavages obtained from 323 patients who were referred to our Clinical Department either for genital complaints or an abnormal PAP smear. We assessed (i) the prevalence of HPV and (ii) the reliability of RFLP-typing. For the latter, 35 PCR-HPV products were sequenced. HPV-DNA was detected in 40/197 (20.3%) patients with normal cytology 86/111 (77.5%) with LSIL and 11/15 (73.3%) with HSIL. HPV-16 was the most common type detected in normal cervical cytology samples (10/40, 25%), whereas HPV 16 and 18 were detected in 36/97 (37.1%) of the LSIL and HSIL patients, evidencing the presence of these high-risk HPV types not only in malignant conditions. Results obtained after partial nucleotide sequencing confirmed the results obtained by RFLP analysis. In this study, a putative new HPV fragment (GA6053) was identified. Its closest homology to other known HPV types is 73.8% to HPV-62, 73.0% to HPV-61 and 67.7% to HPV-18. The use of degenerate primers, in conjunction with RFLP, proved to be a reliable method for HPV detection and typing.

Vertical transmission of hepatitis C virus in low-risk pregnant women.

To assess the prevalence of hepatitis C virus (HCV) infection in pregnant women and the rate of vertical transmission in infected mothers belonging to a low-risk group, 1,388 women were tested for HCV antibody at delivery. Twenty-five anti-HCV-positive women with no apparent source of HCV exposure were recruited. A reverse transcriptase-polymerase chain reaction (RT-PCR) and a new quantitative branched DNA-based signal amplification assay (bDNA) were used to detect HCV RNA. The rate of anti-HCV positivity in pregnant women was 2.5% (36 of 1,388). Of the 25 cohort mothers, 18 (72%) were positive for HCV RNA by RT-PCR, 13 of whom were also positive by the bDNA assay (sensitivity 72.2%). Of the 25 infants of low-risk mothers tested at birth, 22 were anti-HCV positive, two were weakly reactive, one was negative, and none was viremic. Neither active humoral immunoresponse nor HCV RNA was detected in any of the infants over a period of 12 months. These data suggest a relatively high prevalence of anti-HCV in unselected pregnant women and a poor efficiency of vertical transmission of HCV in a low-risk population, irrespective of the viral burden of the mother-to-be.

Detection of HPV-DNA in semen, urine and urethral samples by dot blot and PCR.

Papillomavirus infection in women is associated with the development of carcinoma or squamous intraepithelial lesions (SIL). Limited information is available for men. Seventy asymptomatic male partners of HPV-DNA positive women were studied. Exfoliated cells collected using urethral swabs urine and semen were examined for HPV-DNA using Dot blot and PCR. On exfoliated cells collected using a urethral swab, 89% of the samples were inadequate on Dot blot, and 87% on PCR respectively. Using urine, 36% turned out to be inadequate on Dot blot, 21% on PCR. Using semen all of the 70 samples were satisfactory for both systems. Semen is thus the best material for analysis. The occurrence of HPV-DNA in urine in urine is less frequent than that in semen. Urethral swabs seem to represent the least reliable material. Carriage of human papillomaviruses is frequent in apparently healthy parternrs of HPV infected women.