The comparison of different protocols for expansion of umbilical-cord blood hematopoietic stem cells.

Hematopoiesis is maintained by the activity of multipotent stem cells, which have the dual capacity to self-renew and to differentiate into all of the blood cell lineages. The major challenge of stem cells based regenerative therapy is to expand ex vivo the primitive compartment to increase transplantable stem cells number. The present study was designed to evaluate several culture systems for in vitro maintenance of umbilical cord blood stem cells. The influences of different growth conditions such as stromal feeder layer, cytokines supplement and placental conditioned medium (PCM) have been evaluated over a relatively short period of time on CD34(+) cell expansion and maintenance of clonogenic progenitors. When cells were expanded on feeder layer in the presence of added cytokines and PCM on average a 2.96-fold increase of CD34(+)CD71(-) and a 3.13-fold increase of CD34(+)HLA-DR(-) was observed. The total number of colony forming cells (35 +/- 2.65) indicated also that the yield of clonogenic progenitors obtained with a combination of all factors was two folds higher than each of these factors alone and ten time above control (3.67 +/- 2.52). In conclusion, the results of our study clearly show that the ex vivo expansion of hematopoietic progenitor cells obtained from human umbilical cord blood is dependent on controlled experimental conditions, which might be helpful when designing culture systems for clinical applications.

The development of larger cells that spontaneously escape senescence--a step during the immortalization of a human cancer cell line.

There are few information concerning the changes associated with the transition interval when slow growing, primary explanted human cancer cells are displaced by new selected faster growing cells and became an immortal cell line. In a previous paper (J. Cell. Mol. Med., 5: 49-59, 2001) we described the TV cell line derived from a laryngeal tumor which harbors human papillomavirus (HPV) gene sequences throughout more than sixty in vitro passages. In this paper we analyze the modifications observed during the crisis interval when significant amount of cells senesce but occasional cells acquire some mutations that make them immortal. Confocal microscopy analysis revealed the heterogeneity of the cells in terms of their size and nucleus/cell ratio. Proliferation capacity was assessed by flow cytometry analyzing DNA content and expression of transferrin receptor (CD71). We discussed the possibility that HPV genome sequences alleviate a proliferation block during the crisis growth arrest of human larynx carcinoma cell line and the possibility that the cells monitor their size and growth by measuring the levels of some protein whose synthesis is coupled to cell development.

Ex vivo differentiation of umbilical cord blood progenitor cells in the presence of placental conditioned medium.

Hematopoetic stem cells (HSC) are the progenitors for the lympho-hematopoietic system, with long lifespan and high proliferation potential. Transplantation of HSC from bone marrow or peripheral blood represents a standard therapy in severe hematological conditions. A possible alternative source of HSC is the umbilical cord blood, prepared by various separation procedures followed by expansion in cultures supplemented with hematopoietic growth factors. In order to check the effects of placental conditioned medium (PCM) from placental cells culture upon viability of HSC, we added plasma, PCM, dimetil sulfoxyde or hemin in HSC cultures. Flow cytometry or direct scoring of solid cultures using CD45+, CD34+, CD71+ and CD14+ fluorescent-labeled monoclonal antibodies evaluated the effects upon cell proliferation and colony forming ability of HSC cultures, versus controls. PCM produced the highest proliferation, followed by plasma, DMSO and hemin. PCM improved the survival time and maintained a higher proportion of immature cells. PCM stimulates the differentiation towards myeloid lineage progenitor cells (>90% being CD45+), increasing the percentage of CD14+, granulocites /monocytes precursors. It is highly suggestive that PCM contains growth factors or cytokines, which regulate the development of HSC. Characterization of these factors is in progress.

West nile virus surveillance in Romania: 1997-2000.

In response to the 1996 West Nile (WN) fever epidemic that occurred in Bucharest and southeastern Romania, a surveillance program was established. The surveillance system detected 39 clinical human WN fever cases during the period 1997-2000: 14 cases in 1997, 5 cases in 1998, 7 cases in 1999, and 13 cases in 2000. Thirty-eight of the 39 case-patients lived in the greater Danube Valley of southern Romania, and 1 case-patient resided in the district of Vaslui, located on the Moldavian plateau. The estimated annual case incidence rate for the surveillance area during the period 1997-2000 was 0.95 cases per million residents. Thirty-four cases were serologically confirmed, and 5 cases were classified as probable. Twenty-four case-patients presented with clinical symptoms of meningitis (62%), 12 with meningoencephalitis (31%), 1 with encephalitis (3%), and 2 with febrile exanthema (5%). Five of the 39 cases were fatal (13%). Fourteen case-patients resided in rural areas, and 25 in urban and suburban areas, including 7 case-patients who resided in Bucharest. The ages of case-patients ranged from 8 to 76 years with a median age of 45 years. Twenty-four case-patients were males and 15 were females. Dates of onset of illness occurred from May 24 through September 25, with 82% of onset dates occurring in August and September. Limited entomological surveillance failed to detect WN virus. Retrospective sampling of domestic fowl in the vicinity of case-patient residences during the years 1997-2000 demonstrated seroprevalence rates of 7.8%-29%. Limited wild bird surveillance demonstrated seroprevalence rates of 5%-8%. The surveillance data suggest that WN virus persists focally for several years in poorly understood transmission cycles after sporadic introductions or that WN virus is introduced into Romania at relatively high rates, and persists seasonally in small foci.

Detection of human papillomavirus gene sequences in cell lines derived from laryngeal tumors.

The role of Human Papillomaviruses (HPV) in laryngeal carcinomas has been studied with conflicting results. To evaluate the etiologic relationship between HPV infection and epithelial malignancy of the larynx we studied five laryngeal carcinoma cell lines obtained from patients undergoing surgery for laryngeal tumors. The paraffin embedded biopsy samples of the original tumor and different passages of the new established cell lines were investigated by PCR with consensus primers specific for HPV DNA. The findings indicate that HPV infection is associated with some larynx carcinomas. The positive association has been enhanced when a method of enrichment of epithelial cells from fresh tumor samples was used. All tumor cells enriched smears were positive for HPV DNA not only by PCR but also by in situ hybridization (ISH). Investigated by PCR, different passages of larynx tumor cell lines maintained expression of HPV DNA. At subsequent passages ISH gives constantly no signals suggesting a minimal amount of viral harbored sequences. In one cell line propagated more than 60 population doublings, the chromosomal frequency distribution shifted from modal number 46 at the 5(th) passage to 63 at the 60(th) passage. The mechanisms by which persistent HPV infection maintains continuous cell proliferation were discussed.

Evaluation of immunoglobulin M (IgM) and IgG enzyme immunoassays in serologic diagnosis of West Nile Virus infection.

A unique urban encephalitis epidemic in Romania signaled the emergence of neurological infection due to West Nile (WN) virus as a novel public health threat in Eastern Europe and provided an opportunity to evaluate patterns of immunoglobulin G (IgG) and IgM reactivity in IgM capture and IgG enzyme-linked immunosorbent assays (ELISAs). WN virus infection was diagnosed serologically in 236 of 290 patients from whom acute serum or cerebrospinal fluid (CSF) samples were available. In 37% of serum samples and in 25% of CSF samples collected in the first week of illness, anti-WN virus IgM antibody was detected in the absence of virus-specific IgG. The switch to an IgG antibody response occurred after 4 to 5 days of illness and earlier in CSF than in serum. A specific humoral immune response was detected in the CSF before the serum in some patients for whom paired CSF and serum samples from the same day were available. IgM antibody in convalescent serum samples persisted beyond 2 months after the onset of illness in more than 50% of patients. ELISA optical density values and antibody concentrations were well correlated for both IgM and IgG immunoassays. Anti-WN virus IgM antibody in acute-phase samples did not cross-react significantly with flaviviruses in other antigenic groups.

Phagocytic function of monocytes in children with human immunodeficiency virus type 1 infection.

We investigated the phagocytic function of monocytes in 7- to 10-year-old children horizontally infected with human immunodeficiency virus type 1 (HIV-1) in comparison to that in healthy sex- and age-matched controls. CR3-mediated phagocytosis was increased in patients with HIV-associated pulmonary tuberculosis, independently of CD4 counts and p24 antigenemia.

Continued transmission of West Nile virus to humans in southeastern Romania, 1997-1998.

After an epidemic of West Nile (WN) virus neurologic infections in southeastern Romania in 1996, human and animal surveillance were established to monitor continued transmission of the virus. During 1997 and 1998, neurologic infections were diagnosed serologically as WN encephalitis in 12 of 322 patients in 19 southeastern districts and in 1 of 75 Bucharest patients. In addition, amid a countrywide epidemic of measles, the etiology of the febrile exanthem in 2 of 180 investigated cases was determined serologically to be WN fever; 1 case was complicated by hepatitis. Sentinel chickens placed in Bucharest seroconverted to WN virus during the summer months, indicating their potential value in monitoring transmission. The continued occurrence of sporadic WN infections in southeastern Romania in consecutive years after the 1996 epidemic is consistent with local enzootic transmission of the virus.

Dynamics of the HIV-1 variability in adults from Bucharest, 1992-1998.

Variability is a hallmark of HIV viruses both at the genetic and the phenotypic level. Viral sequencing and phylogenetic analysis of many isolates revealed specific distribution of HIV-1 subtypes according to the geographic location and route of transmission. In Romania, the currently available data coming from the study of pediatric HIV infection suggest the predominance of subtype F. However, there are few data concerning the distribution of HIV-1 subtypes among adults. We investigated the changes in the distribution of different HIV-1 subtypes among HIV-1 infected adult patients from Bucharest over a 6 years period (1992-1998) by means of V3 binding assays. The analysis of the relative incidence of different HIV-1 serotypes revealed the steady predominance of serotype F (50-75%) among the adults from Bucharest and a small but constant increase of the number of samples with serotype B- from 5% in 1994 to 14% in 1998. In contrast, the serotype E is either absent or weakly represented (4%) during the period of the study. All over the years there was a relatively high proportion (6-30%) of non-reactive samples. This could be an indication for the possible emergence of new or recombinant HIV-1 subtypes.

An outbreak of aseptic meningitis due to ECHO 30 virus in Romania during the 1999 summer.

Between April-September 1999 an unusually high number of acute aseptic meningitis was reported in six North Eastern counties of Romania. At the end of 1999 summer the epidemic area extended in South Romania including the Capital City Bucharest. The total number of cases surpassed 5500. The majority of confirmed cases were young children under 15 years of age. More than 1800 cases were registered in the same interval in the neighboring Republic of Moldavia. We report the isolation of ECHO 30 virus from 12 CSF samples of 52 tested on RD and human diploid cells. A small case control study identified high seroneutralization titers confirming a recent ECHO 30 infection in 74% of patient's samples and in 54% of samples from control children tested. The time spent outdoors and poor hygiene practices were prominent risk factors for transmission.

Romanian HIV-AIDS epidemic after a decade of evolution.

Situated in Southeastern Europe, with a surface of 237,500 km2 and a population of over 22 million, Romania was a special case in the evolution of global AIDS crisis. After the first reported case in a homosexual male in 1985, five years elapsed till the epidemiological investigations done in the Institute of Virology, Bucharest (1-4) provided strong evidence for what may represent the world's largest iatrogenic transmission of blood-borne pathogens in children. On the basis of Epidemiological Fact Sheet concerning Romania provided by UNAIDS/WHO Working Group on Global HIV/AIDS and STD Surveillance we offer a couple of graphics which are considered important for informed decision-making and planning at national and regional levels. We emphasize the lack of availability of many specific data both on epidemiological and socio-behavioral indicators. Structural collapse in Eastern Europe sets the scene for the rapid spread of HIV/AIDS among young people and the lack of information may contribute to an unexpected rebound of the epidemic.

Risk factors for West Nile virus infection and meningoencephalitis, Romania, 1996.

In 1996, an epidemic of 393 cases of laboratory-confirmed West Nile meningoencephalitis occurred in southeast Romania, with widespread subclinical human infection. Two case-control studies were performed to identify risk factors for acquiring infection and for developing clinical meningoencephalitis after infection. Mosquitoes in the home were associated with infection (reported by 37 [97%] of 38 asymptomatically seropositive persons compared with 36 [72%] of 50 seronegative controls, P<.01) and, among apartment dwellers, flooded basements were a risk factor (reported by 15 [63%] of 24 seropositive persons vs. 11 [30%] of 37 seronegative controls, P=.01). Meningoencephalitis was not associated with hypertension or other underlying medical conditions but was associated with spending more time outdoors (meningoencephalitis patients and asymptomatically seropositive persons spent 8.0 and 3.5 h [medians] outdoors daily, respectively, P<.01). Disease prevention efforts should focus on eliminating peridomestic mosquito breeding sites and reducing peridomestic mosquito exposure.

West Nile encephalitis epidemic in southeastern Romania.

BACKGROUND: West Nile fever (WNF) is a mosquito-borne flavivirus infection endemic in Africa and Asia. In 1996, the first major WNF epidemic in Europe occurred in Romania, with a high rate of neurological infections. We investigated the epidemic to characterise transmission patterns in this novel setting and to determine its origin. METHODS: Hospital-based surveillance identified patients admitted with acute aseptic meningitis and encephalitis in 40 Romanian districts, including Bucharest. Infection was confirmed with IgM capture and indirect IgG ELISAs. In October, 1996, we surveyed outpatients in Bucharest and seven other districts to estimate seroprevalence and to detect infected patients not admitted to hospital. We also measured the rates of infection and seropositivity in mosquitoes and birds, respectively. RESULTS: Between July 15 and Oct 12, we identified 393 patients with serologically confirmed or probable WNF infection, of whom 352 had acute central-nervous-system infections. 17 patients older than 50 years died. Fatality/case ratio and disease incidence increased with age. The outbreak was confined to 14 districts in the lower Danube valley and Bucharest (attack rate 12.4/100000 people) with a seroprevalence of 4.1%. The number of mild cases could not be estimated. WN virus was recovered from Culex pipiens mosquitoes, the most likely vector, and antibodies to WN virus were found in 41% of domestic fowl. INTERPRETATION: The epidemic in Bucharest reflected increased regional WNF transmission in 1996. Epidemics of Cx pipiens-borne WNF could occur in other European cities with conditions conducive to transmission.

"Trial moves rapidly on, when the judge has determined the sentence beforehand" (W. Scott: Ivanhoe, 36) or pitfalls in serosurvey of anti-hepatitis C antibody in children.

Hepatitis C is and will be a major public health concern. Confirmed infections were reported from all Romanian counties but important differences between regions raise several explanations. Differences may reflect the different levels of testing, the performances of laboratories in confirming initially reactive samples or the risk factors higher prevalence. We have suggested that the prevalence of anti HCV infections can be a surrogate marker for the quality of parenteral medical or paramedical interventions. Present report identified additional problems in the surveillance of HCV infection in children. We screened 1787 samples from children hosted in orphanages (children under three years old) or in preschool children institutions (between 3-7 years old). We detected 31 repeatedly reactive samples with two EIA screening kits but confirmed only 8 in WB anti HCV. Four confirmed samples come from children under four months old suggesting maternally transmitted antibodies. In highly endemic area, many infants have maternally derived antibodies and the wane of reactivity comes with age above 12 months. Therefore, the prevalence of anti HCV antibody in infants reflects the prevalence in adult population. Confirmatory tests are mandatory for the serosurvey in children. More frequent than adults samples, children EIA reactive samples give indeterminate or negative Western Blot profiles. Only the viral load evaluation can confirm those samples as false positive or, on the contrary, samples at the beginning of seroconversion.

Assessment of the anti-hepatitis B vaccination efficacy in high risk children.

In October 1995, The Ministry of Health has initiated the national immunization program of newborns against hepatitis B. Owing to the frequency of asymptomatic Hepatitis B clinical forms in children, as well as the deficiencies in the surveillance system, the assessment of the vaccination efficacy can be performed objectively only by the detection of the prevalence of anti HBs antibodies in children to whom the complete three doses of immunization schedule have been administered (at 0, 2 and 6 months of age). We report in this study the results of a seroprevalence research carried out on a group of 272 children from orphanages who have been vaccinated. A protective anti HBs titer (> 10 mIU) was recorded only in 66.3% of cases; other 10 samples contained antibodies at a titer lower than the protective level. In the 80 children without seroconversion the presence of anti HBc antibodies (marker for the natural infection) was investigated. 30% of the seronegative children have anti HBc antibodies from which 54.2% have also HbsAg. Significant differences were recorded in the seroconversion level and in the geometric mean of titers between the various units in which sera were collected. In four orphanages (district Arad, Jassy, Sibiu and Teleorman) the seroconversion exceeded 90%, in 5 orphanages it was over 80% and in the others it ranged from 30% to 70%. The lowest seroconversions were recorded in the orphanages in Bucharest, Botosani, Galati and Olt. The possible causes of the low immunogenicity are analyzed: non-vaccination or incomplete vaccination; low immunoreactivity of children, many of whom are premature; high HbsAg carriage rate among the mother's etc. Although the evolution of the post vaccinal seroconversion is not a routine practice in the appraisement of Hepatitis B vaccine immunogenicity, our results require the extension of the study in order to adopt the most effective vaccinal strategy.

Cerebrospinal fluid western Blot profiles in the evolution of HIV-1 pediatric encephalopathy.

In order to obtain information on neurologic AIDS, 54 white caucasian children infected by nosocomial route with a median age of 46.2 +/- 7 months were followed up prospectively for a median of 12 months with three months Denver tests neurologic evaluation and six months serologic investigations in CSF and sera. Paired CSF and serum samples, collected on the same day, from children with AIDS encephalopathy, were analysed for the permeability of the blood brain barrier (BBB) and for intrathecal production of anti HIV specific antibodies. A prospective follow-up and repeated comparison of WB profiles and the presence of anti V3 antibodies in CSF and sera was done, as well as an evaluation of the modification in the CSF antibody specificity (anti gag Western Blot scoring) with disease progression. An increased intrathecal synthesis of IgG was recorded in all subjects, in spite of an unaltered BBB permeability. No significant differences were recorded for the anti gag score in the serum samples, which was stable between 9.1-10.4. By contrast, the score for CSF samples decreases significantly with disease progression, from 8.7 in children without encephalopathy, to 6.5 in those with stationary disease and 3.6 in the progressive encephalopathy group. A strong correlation was found between the level of anti p24 antibodies determined by ELISA and the anti gag score quantified by WB for the same CSF samples. The p24 antigen was found to be positive only in 3 cases, even after immune complex dissociation. Anti V3 antibodies were not detected in CSF samples from patients with functional BBB. The decline in anti-gag antibody reactivity in CSF is an early indicator of disease progression, reflecting a severe course of neurological impairments. The absence of anti V3 antibodies in the CSF samples suggests that the PND of neurotropic strains mapped in distinct positions into the V3 loop. These results reflect the selection of antigenic escape mutants which evolve in the CNS, distinct from the blood lymphotropic isolates.

Low rates of seroconversion after hepatitis B vaccination in orphanges with high prevalence of virus carriers.

A serosurvey of Hepatitis B infection markers was conducted in two orphanages that adhered to Hepatitis B vaccination policy. In spite of comparable sizes (80-90 children per facility), housing conditions and infection control practices, the level of HbsAg endemicity was different in each unit in direct relation with the mean age of the children. The prevalence of HbsAg carriers and the interval spent in collectivity strongly affect the seroconversion rate after HB vaccination. Other elements that can explain the low seroconversion rate were: the proportion on fully vaccinated children, the number of vaccine administered doses and the delayed age at which childhood immunization schedule was initiated. In order to increase the protective antibody response, booster doses were administered to a limited number of nonseroconvertors or to children with a nonprotective level of anti-HBs antibody (< 10 UI). This intervention provides evidence of prompt rising in antibody titers, comparable with titers found in children with wild infection.

A high number of severe neurologic clinical forms during an epidemic of West Nile virus infection.

The serologic confirmation of more than 800 cases hospitalized during the viral meningoencephalitis epidemic caused by the West Nile virus (WNV) that affected the South-East of Romania during the summer of 1996 consolidated the case definition in over 80% of the patients admitted to the hospital with neurological impairments. Other clinical forms of the WN infection were reported only scarcely during the epidemic and were seroconfirmed at a lower rate (60%). IgM capture ELISA (MAC-ELISA) is a test of choice for the rapid diagnosis. The major advantage of MAC-ELISA procedure is the high probability of accurate diagnosis of WN infection when the test is performed only with acute serum or cerebrospinal fluid (CSF) specimens obtained while the patient is still hospitalized. Rapid diagnosis by MAC-ELISA is important for the institution of public health control, but the results obtained have also some predictive values. We report the serological patterns of 65 pairs of CSF and serum samples collected in the early days of neuroinfection for diagnostic purposes. An unexpected onset of the intrathecal specific humoral immune response before serum immunoglobulins synthesis was recorded in 25% of cases. For 14 patients with intrathecal onset of IgM synthesis, their records evaluated retrospectively showed a severe evolution. The presence of only IgM antibodies in CSF is a characteristic which matched with other laboratory variables described which predict poor evolution in viral encephalitis: pleocytosis, elevated protein concentration in CSF (> 100 mg per deciliter), hyponatremia (< 130 mmol per liter).

Serological profile assessment of the infection with hepatitis C virus (HCV) in haemophiliacs and thalassemic patients.

Polytransfused patients represent a major risk group for hepatitis C (HCV) acquirement. Haemophiliacs and thalassemic patients treated with virus contaminated blood or blood derivatives frequently exhibit anti-HCV antibodies and signs of chronic hepatitis. The serological profile for the HCV infection was investigated in 13 haemophiliacs, 18 cases of thalassemia and in 14 polytransfused patients affected by other diseases. The presence of anti-HCV antibodies was detected by means of the ORTHO HCV 3.0 ELISA kit and confirmed by Western-blot Murex. The serotyping used synthetic peptides mimicking the immunodominant epitopes in the NS4 region, characteristic of each of the six HCV genotypes in an ELISA blocking reaction (Murex). Serotype 1 was prevalent (51.1%), while serotype 2 was detected in 13.3% of patients, with a higher frequency in thalassemia cases. The remaining samples were multireactive, and serotype 3 alone was not detected. The profile of Western-blot bands was distinct for the monoreactive samples belonging to serotype 1 or 2. The analysis of the multireactive samples in young (thalassemic, age mean 15.17 +/- 6.5) and aged patients (haemophiliacs, age mean 32.64 +/- 13.5) allows us to suggest a different succession of reinfection acquirements. The infection with one of the subtypes does not confer protection against the reinfection with others. However, a certain attenuation of the symptomatology is obvious in the case of reinfections, indicating the existence of crossantigenic reactivities which contribute to protection. This protection is more evident in the case of primary infection with type 2 and is partially due to antigens coded by the NS4 genomic segment.

Immunological disorders of increased severity in children with AIDS associated with hepatitis B and C infections.

Two groups of children were set up: children infected with HIV and hepatitis viruses and children infected with HIV, but not with hepatitis viruses, too. The immunological aspects investigated referred to the Ig serum value, the absolute number of T CD4 lymphocytes and the T CD4/T CD8 ratio. The subjects of the first group (in whom hepatitis markers were present) displayed increased IgA and IgM values at a higher rate than those of the second group (54.87% against 32%, 83.3% against 53%), as well as a lowering below 300/ml of the number of T CD4 lymphocytes (50% against 29.4%). On the other hand, increased IgG levels and values below 0.8 of the T CD4/T CD8 ratio were found at similar rates in the two groups of children (77.4% against 80% and 70.5% against 70% respectively). By means of the data obtained, the authors try to point out one of the ways by which hepatitis viruses, considered as a potential cofactor in the AIDS development, contribute to the course of this disease, namely by intensifying the immunological disorders.