A safer plasma supply from remunerated donors--"The Immuno/Community Bio-Resources experiment".

With the goal of increasing the safety of plasma used in the manufacture of therapeutic products, Immuno and its subsidiary Community Bio-Resources (now a division of Baxter Healthcare Corporation), have developed a comprehensive plasma quality programme. This programme includes four main safety initiatives: a plasma centre location/appearance programme, a Qualified Donor programme, an Inventory Hold, and the PCR testing of plasma pools. Many of these initiatives have been adopted in part by the plasma collection and fractionation industry. Using a statistical model that takes into consideration the unique donation characteristics of remunerated plasma donors, combined with 1998 CBR virus reactive rates, an estimated residual likelihood of an undetected donation entering a plasma pool was determined. These estimates, for each million donations, were 0, 1.64, and 4.68 donations for HIV, HBV, and HCV, respectively, and were far below those previously reported for remunerated or volunteer donations. These estimates were confirmed by subsequent PCR testing, which allowed for the additional removal of positive units before manufacture. The low virus load of this plasma supply, combined with increasingly effective virus removal and inactivation procedures, has resulted in the safest ever supply of plasma derivatives.

CUS2, a yeast homolog of human Tat-SF1, rescues function of misfolded U2 through an unusual RNA recognition motif.

A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.

Conservation of structure and subunit interactions in yeast homologues of splicing factor 3b (SF3b) subunits.

Human SAP 49, a subunit of the multimeric splicing factor 3b (SF3b), contains two RNA recognition motifs (RRMs) and binds another SF3b subunit called SAP 145, whose yeast homologue is CUS1. Here we show that the predicted yeast open reading frame YOR319w (HSH49) encodes an essential yeast splicing factor. Using bacterially expressed proteins, we find that yeast HSH49 binds CUS1. Mutations that alter putative RNA-binding residues of either HSH49 RRM are lethal in vivo, but do not prevent binding to CUS1 in vitro, suggesting that the predicted RNA-binding surfaces of HSH49 are not required for interaction with CUS1. In vivo interaction tests show that HSH49 and CUS1 associate primarily through the N-terminal RRM of HSH49. Recombinant HSH49 protein has a general RNA-binding activity that does not require CUS1. The parallels in structure and interaction between two SF3b subunits from yeast implies that the mechanism of SF3b action is highly conserved.

[Plasma donor screening and product safety]. / Plasmaspenderscreening und Produktsicherheit.

In addition to the specific virus reduction and inactivation procedures during the manufacturing process, the thorough selection of the source material is an essential factor for the product safety of plasmaderivatives. Screening of donors, inventory hold, and plasma pool testing by PCR are important aspects in raising the safety margin. Between January 1, 1994 and June 30, 1996, 576,673 plasma donations were collected in the German and Austrian plasmapheresis centers of IMMUNO. Incidence rates for viral markers (confirmatory tests) as antibodies against HIV, HCV, and HB surface antigen (HBsAg) were calculated as 0.35, 0.87, and 0.87 per 10(5) donations, respectively. Due to look-back, 1.33% of the donations were eliminated between January and October 1995. In 32% of the donations being rejected in the time of inventory hold in case of elevated alaninamino-transferase (ALT) of the donors, HCV genomes were shown which emphasizes the importance of this surrogate marker. Out of 1,240 pilot pools tested by quality assured PCR (IQ-PCR) being performed by IMMUNO since October 1995, in 4% of cases HCV and in 0.2% HBV genomic equivalents were shown. However, genomes of HIV were not found. The measures aiming at a safe plasma pool are part of IMMUNO's safety concept which is confirmed by the fact that there was no transmission of hepatitis or AIDS viruses by IMMUNO products, since virus inactivation procedures have been established.

RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site.

A yeast gene homologous to bacterial RNase III (RNT1) encodes a double-strand-specific endoribonuclease essential for ribosome synthesis. Two rRNA processing events are blocked in cells temperature sensitive for RNT1: cleavage at the snoRNA-dependent AO site in the 5' ETS and cleavage in the 3' ETS. Recombinant RNT1 protein accurately cleaves a synthetic 5' ETS RNA at AO site in vitro, in the absence of snoRNA or other factors. A synthetic 3' ETS substrate is specifically cleaved at a site 21 nt downstream of the 3' end 28S rRNA. These observations show that a protein endonuclease collaborates with snoRNAs in eukaryotic rRNA processing and exclude a catalytic role for snoRNAs at certain pre-rRNA cleavage.

CUS1, a suppressor of cold-sensitive U2 snRNA mutations, is a novel yeast splicing factor homologous to human SAP 145.

The function of U2 snRNA in splicing is mediated by the proteins of the U2 small nuclear ribonucleoprotein. To identify proteins that influence the function of U2 snRNA we carried out a screen for mutations in Saccharomyces cerevisiae that suppress the cold-sensitive growth defect of a mutation in U2 stem loop IIa, a structure important for the stable association of the U2 snRNP with pre-mRNA. The screen identified three dominant suppressor genes, one of which, CUS1-54, encodes an essential splicing protein required for U2 snRNP addition to the spliceosome. The suppressor protein rescues the spliceosome assembly defect of the mutant U2 in vitro, indicating that suppression is direct. Allele specificity tests show that the suppressor does not simply bypass the requirement for U2 stem loop IIa. Extra copies of wild-type CUS1, but not CUS1-54, suppress the temperature-sensitive prp11 and prp5 mutations, linking CUS1 protein to a subset of other factors required at the same step of spliceosome assembly. CUS1 is homologous to SAP 145, a component of the mammalian U2 snRNP that interacts with pre-mRNA. The yeast genome also encodes a homolog of human SAP 49, a protein that interacts strongly with both SAP 145 and pre-mRNA, underscoring the conservation of U2 snRNP proteins that function in spliceosome assembly.

Histologic analysis of placental tissue in first trimester abortions.

The value of histologic evaluation in the analysis of material from first trimester abortions is not completely defined. We prospectively analyzed placenta and decidua from 75 first trimester, spontaneous abortions to ascertain if morphologic features were predictive of karyotype. The histologic features analyzed included hydropic villus change, villus fibrosis, villus scalloping with trophoblastic invaginations, atypical stromal cells, aggregates of lymphocytes in placenta or decidua, and acute inflammation of placenta or decidua. Normal karyotypes were observed in 44 cases and abnormal karyotypes were demonstrated in 31. The presence of villus scalloping with trophoblastic invagination was significantly associated with abnormal karyotypes, particularly triploidy, and the demonstration of acute inflammation was seen significantly more often in cases with normal karyotypes. We conclude that histology can provide only a suggestion as to the likelihood of an abnormal karyotype; the findings are not specific enough to obviate the need for karyotyping in the individual case.

PIP: Placenta and decidua from 75 1st-trimester spontaneous abortions were prospectively analyzed to determine whether morphologic features were predictive of karyotype. The histologic features analyzed included hydropic villus change, villus fibrosis, villus scalloping with trophoblastic invaginations, atypical stromal cells, aggregates of lymphocytes in placenta or decidua, and acute inflammation of placenta or decidua. Of the 103 cases submitted during the 12-month study period, 75 had successful karyotypes and sufficient histologic material for analysis. Normal karyotypes were found in 44 cases (26 females and 18 males). The 31 cases with abnormal karyotypes included 10 cases of triploidy, 9 cases of trisomy, 6 cases of monosomy, 3 cases of tetraploidy, and 3 cases of unbalanced translocation. The presence of villus scalloping with trophoblastic invaginations was seen significantly more frequently in cases with abnormal karyotypes (p0.05). The positive predictive value of this finding was 59% and the negative predictive value was 75%. Analysis of specific karyotype abnormalities demonstrated that triploid cases contributed the majority of cases with these villus changes. Acute inflammation of the placenta and decidua was significantly associated with a normal karyotype (p0.01). A low frequency of acute inflammation was observed in all the specific karyotype abnormalities. The other histologic features analyzed were found with approximately equal frequency in placentas with normal and abnormal karyotypes. These findings indicate that histology can provide only a suggestion as to the likelihood of an abnormal karyotype; the results are not specific enough to obviate the need for karyotyping in the individual case.

Evaluation of a latex particle agglutination kit in pneumococcal disease.

Latex particle agglutination for Streptococcus pneumoniae was evaluated in 76 patients. Fifteen of these patients had invasive disease due to S. pneumoniae including 12 with meningitis, 2 with occult bacteremia and 1 with suppurative arthritis. Five of the patients with meningitis also had bacteremia. Pneumococcal antigen was detected in the cerebrospinal fluid of 9 of the 12 patients with meningitis (sensitivity 75%). However, antigen was detected in the serum of only two of the six patients with bacteremia (sensitivity 33%) and was detected in the urine of none of five patients with bacteremia (sensitivity 0%). Consequently latex particle agglutination appears to be useful when cerebrospinal fluid is examined in patients with pneumococcal meningitis but does not appear to be sufficiently sensitive to warrant its use with serum or urine in patients with invasive disease due to S. pneumoniae. The specificity of the system used here appeared satisfactory, since pneumococcal antigen was not detected in any of the body fluids from the 61 patients without evidence of invasive pneumococcal disease (specificity 100%).

Chemical carcinogen induction of DNA-repair synthesis in human peripheral blood monocytes.

Fresh ex vivo cultures of normal human peripheral blood monocytes, which are nonreplicative and known to possess cytochrome P-450 associated mixed-function oxidase activity, were used to assay DNA-excision repair manifested as augmented [3H]thymidine (dThd) incorporation following treatment in culture with diverse mutagenic carcinogens. Untreated monocyte cultures established from pools of 3-6 normal donors incorporated a low level of cytoplasmic [3H]dThd throughout a majority of the cells during an 18-h incubation. This background incorporation into whole cells was 80-90% inhibited by hydroxyurea (HU) at concentrations greater than 5 mM. Dose-related increases in the cumulative 18-h [3H]dThd incorporation in monocytes were observed following treatment with UV, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), mitomycin C (MMC), N-acetoxy-acetylaminofluorene (NA-AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), and dimethylnitrosamine (DMN). The presence of HU during chemical treatment and throughout this 18 h of incubation with [3H]dThd did not influence the dose-response curves obtained with UV, MMS, NA-AAF and BaP but it increased the input dose of MNNG, MMC, DMN and AFB1 required to give peak repair incorporation. When HU was added to cultures following MNNG damage no interference with repair response was observed. HU apparently influences the extent of DNA damage by direct reactivity with these chemicals or their endogenously generated metabolites rather than inhibiting DNA-repair processes. These results provide evidence that monocytes are enzymatically proficient in base and nucleotide excision pathways and have endogenous capacity to metabolize BaP, AFB1 and DMN to DNA-damaging metabolites. As such, the monocyte is a potentially useful human cell type for detecting genotoxic chemicals and studying individuality in chemical-biological interactions.

[Carcinoma after-care in an after-care clinic based on experiences at the Sonnenberg Clinic, Bad Soden-Allendorf]. / Probleme der Krebsnachsorge in einer Krebsnachsorge-Klinik am Beispiel der Sonnenberg-Klinik Bad Sooden-Allendorf.

The follow-up cure therapy in the framework of cancer follow-up treatment is of special importance. During the past 7 years 7814 patients have been treated according a special regimen in our clinic, which also includes cytostatic and hormonal therapy. Diagnostic control examinations for all patients are performed. Besides adequate medical treatment psychological and social aspects of the individual patient should be considered, furthermore the course of the disease, the question of again taking up life with their families and their partners and of returning to their former professional work. It could be shown that general discussion groups and cancer selfhelp groups, which are organized by the patients, are fruitful. It seems beneficial for the patients to participate in these activities in a follow-up clinic for a course of 6 weeks and they seem to recuperate better regarding their general health and survival time.

Heteroploid conversion of human skin cells by methylcholanthrene.

Cultured epithelial cells from human skin generally had 3- to 30-fold more hydrocarbon-metabolizing activity than fibroblasts from skin of the same donor. This activity was constant for up to 55 days in primary culture but was lost rapidly upon physical subdivision of the cultures. Treatment of primary mixed fibroblasts and epithelial cell cultures with methylcholanthrene, but not phenanthrene, led to development of actively growing fibroblastic cultures with many heteroploid cells. Unique marker chromosomes, stable over a number of cell population doublings, were identified in several of the heteroploid cell strains. Pure cultures of fibroblasts from the same donors did not undergo heteroploid conversion in response to methylcholanthrene. Spontaneously occurring heteroploidy in logarithmic phase human fibroblasts is a rare event; thus, heteroploid conversion may be a useful marker for chemical transformation of human cells. Because methylcholanthrene seems to have little transforming effect on human skin fibroblasts, human skin epithelial cells, because of their hydrocarbon-metabolizing activity, may serve to convert methylcholanthrene from a distal to an ultimate carcinogenic form.

Growth and characterization of human skin epithelial cell cultures.

In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epilthelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis.

Lymphocytic choriomeningitis virus infection in fetal, newborn, and young adult Syrian hamsters (Mesocricetus auratus).

The pathogenesis of lymphocytic choriomeningitis virus infection in fetal, newborn, and young adult hamsters was studied. Infected newborn hamsters initially developed a persistent viremia and viruria with titers often in excess of 10(4.0) mean infectious doses/0.03 ml of blood or urine. After week 12 two different patterns of infection became evident. Approximately one-half of the hamsters eventually cleared the infection, whereas the others developed a chronic progressive and ultimalely fatal disease characterized by continuous high-titered viremia and viruria and high titers of virus in their tissues. Complement-fixing antibody and, to a lesser degree, virus-neutralizing antibody coexisted with the viremia. Hamsters with persistently high levels of viremia and viruria developed chronic glomerulonephritis and widespread vasculitis, whereas hamsters that cleared their infections did not develop these lesions. Litters of hamsters born to viremic mothers were invariably infected. Litter sizes were small and breeding effectiveness was reduce; however, vertical, congenital infection was successfully passed through three generations. The course of infection in the congenitally infected hamsters was similar to that in newborn infected hamsters, with all animals producing complement-fixing antibody, some animals being capable of clearing the viremia and remaining healthy, and other animals having persistent viremia and fatal disease. Inoculated young adult hamsters did not become diseased, developed viremia and viruria which persisted up to 3 and 6 months, respectively, and developed complement-fixing antibody by 10 days after infection. The prolonged urinary excretion of large amounts of lymphocytic choriomeningitis virus by asymptomatic, chronically infected hamsters is an important public health consideration when dealing with potential human infection.

Carcinogenesis in vitro. II. Chemical transformation of diploid human cell cultures: A rare event.

Seventy-five diploid human cell s-rains were subjected to a number of chemical carcinogens, including urethane and polycyclic hydrocarbons. In most cases, no visible morphological alterations were induced by any treatment. Development of morphologically altered foci was noticed in urethane-treated cultures derived from a patient with von Recklinghausen's disease. This disease is transmitted by an autosomal dominant gene, and has a high rate of spontaneous transformation of neurofibromas to neurofibrosarcomas. Attempts to isolate continuous cell lines from altered foci were successful in only two of several attempts. These continuous cell lines demonstrate altered morphology, loss of contact inhibition, accelerated growth rate, and have attained over 240 generations in a period of 140 weeks. Untreated control cultures became terminal by the 20th generation. Giemsa banding procedures showed that the chromosomal complement consisted of heteroploid human chromosomes. A second diploid cell strain derived from the above patient's sibling, also suffering from von Recklinghausen's disease, likewise was morphologically altered by urethane. Chemical transformation of human cells is difficult to induce; however, selection of genetically predisposed cells and prolonged, intermittent, and repeated chemical treatment may be important factors in achieving transformation.

I. In vitrol transformation of rat embryo cells: correlations with the known tumorigenic activities of chemicals in rodents.

Susceptibility to chemically induced transformation changed as a rat embryo cell culture was passaged. For the first 35 to 60 passages, the cultures were diploid and resistant to transformation by chemical carcinogens. However, cultures infected with a murine leukemia virus were transformed by chemicals. For the next 60 passages, the cultures were heteroploid, but retained contact inhibition and were not tumorigenic. Even without addition of heterotypic viruses, these heteroploid cultures could be transformed by chemicals, but the endogenous rat C-type virus could be demonstrated in the transformed cultures. At higher passages, the rates of spontaneous transformation gradually increased so that the cultures could not be used for transformation studies. Chemically induced transformation of the stable heteroploid cell line (F1706) was manifested by an easy to read focal alteration. Initial observations based on these foci were confirmed by inoculating the morphologically altered cells into isogeneic newborn rats. A number of carcinogenic and noncarcinogenic chemical analogues were tested for their ability to transform F1706 cultures. The compounds tested included 4 azo dyes, 12 polycyclic hydrocarbons, 12 aromatic amines, and 7 miscellaneous compounds. Based on the known activities of the same chemicals in rodents, certain active compounds failed to induce transformation in any test, and others induced transformation in only some tests, but these in vitro tests, if used as a screening assay, would have been correct in 82% of all individual tests, and over-all, would have correctly predicated the carcinogenic activity of 33 of the 35 agents tested.