Tolerance induction by costimulator blockade in 100 cGy treated hosts with varying degrees of genetic disparity.

Long-term multilineage allochimerism can be obtained in H2-mismatched B6.SJL to BALB/c transplants with host irradiation of 100 cGy, donor spleen cell pre-exposure and costimulator blockade with anti-CD40 ligand (CD40L) antibody. We evaluated this allochimerism approach in murine marrow transplants with different degrees of major histocompatibility complexe (MHC) mismatching; these include: (1) H2-mismatched transplant H2Kk to H2Kb, (2) full haplo-identical transplant H2Kbd to H2Kbk, (3) a partial haplo-identical transplant H2Kd to H2Kbd and (4) an MHC class II mismatch. Levels of chimerism increased up to 12 weeks and then stayed relatively stable up to 1 year after transplant. At 18 weeks post-transplant, the H2-mismatched, haplo-identical, partial haplo-identical and class II-mismatch transplants evidenced 17.9+/-4.4, 40.7+/-0.9, 25.1+/-4.19 and 33.7+/-3.5% donor chimerism, respectively. Dropping the anti-CD40 antibody treatment and spleen cells or changing the schedule of antibody to one injection, in haplo-identical or full-mismatched transplants resulted in no donor-derived chimerism. On the other hand, these still resulted in minor chimerism in class II-mismatched transplants. Lineage analysis of peripheral blood at 6 and 12 months post-transplant demonstrated a significant shift toward increased chimeric lymphocytes and decreased chimeric granulocytes in the full H2 as compared with haplo-identical or class II transplants. Transplantation with anti-CD40L antibody eliminated both graft-versus-leukemia and graft-versus-host disease (GVHD) and delayed lymphocyte infusion did not rescue animals from fatal leukemia. In conclusion, under the conditions of our tolerization regimen, a haplo transplant gives higher engraftment levels than a full H2 mismatch, and despite lower engraftment levels, a class II-mismatched transplant can be successfully accomplished with only 100 cGy and no CD40L blockade.

The adult hemopoietic stem cell plasticity debate: idols vs new paradigms.

The debate surrounding adult stem cell plasticity derives from a confusion regarding definitions of important terms and the identification of key questions. After defining plasticity, lineage, differentiation and transdifferentiation, the authors put forth a framework for future dialogue as well as their perspective on the stem cell plasticity debate.

p53: a good diagnostic marker for intratubular germ cell neoplasia, unclassified.

Intratubular germ cell neoplasia, unclassified (IGCNU) is a precursor of germ cell tumors (GCT) of the testis. In routine histologic sections, neoplastic intratubular germ cells may be very few and easily overlooked. The aim of this study is two-fold: to establish the immunohistochemical pattern of expression of p53 in IGCNU and GCT and to determine whether p53 can be used as a marker for IGCNU. Resection specimens from 14 seminomas, 14 mixed germ cell tumors (MGCT), 3 embryonal carcinomas, 2 mature teratomas, 7 IGCNUs, and 11 normal testes were stained for p53. Normal germ cells and Sertoli cells of the seminiferous tubules in all normal testes were negative for p53. The tumor cells of all IGCNU cases were positive for p53. All invasive components of mixed germ cell tumors, embryonal carcinomas, and seminomas exhibited expression of p53. Mature teratoma components were negative for p53. These findings indicate that p53 is a highly sensitive marker of IGCNU and highly specific in distinguishing lesional tissue from normal seminiferous tubules. The current findings also suggest that p53 may be involved as an early step in the malignant progression of most germ cell neoplasias.

Major histocompatibility complex class I-recognizing receptors are disease risk genes in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a heterogeneous syndrome of which a subset of patients develops vascular inflammation. The genetic determinants that confer risk for rheumatoid vasculitis are not known, but patients with vascular complications are known to have an expansion of CD4(+)CD28(null) T cells, a cell population potentially involved in endothelial damage. CD4(+)CD28(null) T cell clones isolated from RA patients with vasculitis were found to express killer cell immunoglobulin-like receptors (KIRs) with the stimulatory KIR2DS2 often present in the absence of opposing inhibitory receptors with related specificities. To test the hypothesis that the KIR2DS2 gene is involved in the development of vasculitis, association studies were performed. The KIR2DS2 gene was significantly enriched among patients with rheumatoid vasculitis compared with normal individuals (odds ratio 5.56, P = 0.001) and patients with RA but no vasculitis (odds ratio 7.96, P = 0.001). Also, the distribution of human histocompatibility leukocyte antigen (HLA)-C, the putative ligand for KIRs, was significantly different in patients with rheumatoid vasculitis in comparison with the control populations. These data suggest that HLA class I-recognizing receptors and HLA class I genes are genetic risk determinants that modulate the pattern of RA expression. Specifically, KIR2DS2 in conjunction with the appropriate HLA-C ligand may have a role in vascular damage by regulating CD4(+)CD28(null) T cells.

Nonprofit to for-profit hospital transactions: all roads lead to the attorney general's office.

As more and more hospitals travel the route from nonprofit to for-profit status, state attorneys general are increasingly playing the role of "traffic cop" along this rough and often contentious road. A better understanding of the attorney general's office and greater rapport with its officers is the "order of the day" for officers and directors looking to orchestrate such a transition.

Treatment of antidepressant-induced sexual dysfunction.

Sexual dysfunction is a common side effect seen in patients taking selective serotonin reuptake inhibitors. Spontaneous resolution may occur in some patients, but modifications to therapy can be employed to eliminate undesirable side effects. These include reducing drug dosages, altering timing of drug dosages, taking drug holidays, adding an adjunctive drug, and switching to alternative antidepressants.

A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production.

We have isolated a naturally arising human immunodeficiency type 1 (HIV-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominant production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced messages contributed to reduced virus replication as a result of a reduction in wild-type Rev protein. The point mutation within exon 6D activated exon 6D inclusion when the exon and its flanking splice sites were transferred to a heterologous minigene. Introduction of the point mutation into an otherwise wild-type HIV-1 proviral clone resulted in virus that was severely inhibited for replication in T cells and displayed elevated usage of exon 6D. Exon 6D contains a bipartite element similar to that seen in tat exon 3 of HIV-1, consisting of a potential exon splicing silencer (ESS) juxtaposed to a purine-rich sequence similar to known exon splicing enhancers. In the absence of a flanking 5' splice site, the point mutation within the exon 6D ESS-like element strongly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing. We propose, therefore, that exon silencers may be a common element in the HIV-1 genome used to create balanced splicing of multiple products from a single precursor RNA.

Effects of diet and hindgut defaunation on diet digestibility and microbial concentrations in the cecum and colon of the horse.

The effects of diet and hindgut defaunation (removal of protozoa from the hindgut) on diet digestibility (Trial 1) and on total and cellulolytic bacterial and fungal concentrations in the cecum and colon (Trial 2) were investigated. A high-forage (HF) diet, 90% alfalfa hay-10% concentrate, or a higher-concentrate (HC) diet, 60% alfalfa hay-40% concentrate, was limit-fed. In Trial 1, defaunation resulted in a slight decrease in DM digestibility (P < .1) and had no effect on cellulose digestibility. Dry matter digestibility was higher (P < .001) with the HC diet; however, no differences were observed in cellulose digestion. For the faunated periods, protozoal concentrations were similar in the cecum and greater in the colon for both diets (P < .05). A diet x location interaction was observed for the genera Buetschlia and Blepharocorys. In Trial 2, defaunation had no effect on either total or cellulolytic bacterial concentrations in the cecum or colon. Total bacterial concentrations were higher (P < .06) in the colon when ponies were fed the HC diet. Defaunation did not affect total fungal concentrations in the cecum; however, fungal concentrations in the colon were slightly higher (P < .1) when the ponies were defaunated. Diet had no effect on total or cellulolytic fungal concentrations. Both total and cellulolytic fungal concentrations were approximately 10-fold higher in the colon than in the cecum (P < .01). Protozoa do not seem to play an essential role in the fermentation of feedstuffs in the equine hindgut.

Dietary clofibric acid increases intestinal fatty acid binding protein activity and apparent lipid digestibility in weanling swine.

The effects of supplementation of dietary clofibric acid (.5% wt/wt) on fatty acid binding protein (FABP) activity, apparent lipid digestibility, and serum cholesterol concentrations were evaluated in weanling pigs. Twenty-four barrows were allotted by weight and litter to a randomized complete block design with two treatments (basal vs clofibric acid) in six replicates. Nutrient digestibility measurements were made for a 2-wk period, after which the pigs were killed and tissues were collected. No differences in BW, liver, proximal small intestine, distal small intestine, and proximal and distal intestinal mucosa weights were observed. Apparent lipid digestibility was greater (P < .05) for the overall 2-wk period in clofibric acid-supplemented pigs (81.5 vs 76.6%). This paralleled the increased FABP activity in the distal small intestine (P < .001) of clofibric acid-supplemented pigs. Proximal intestine and liver FABP activities were unaffected by dietary treatment. Serum cholesterol concentrations were markedly lowered by clofibric acid supplementation. During wk 1, pigs fed the basal diet had twofold greater (P < .01) serum cholesterol concentrations, whereas during wk 2, basal-fed pigs had fourfold greater (P < .01) serum cholesterol concentrations (81.5 vs 18.3 mg/dL). These results suggest that elevated intestinal FABP activities may augment fatty acid absorption from the gastrointestinal tract.

HIV recovery from saliva before and after dental treatment: inhibitors may have critical role in viral inactivation.

Unstimulated whole saliva was collected from 21 HIV-positive men and women before and after dental treatment. The frequency of HIV detection did not increase after dental treatment. Infectious HIV was recovered from only one patient. Study findings raise the possibility that, in most cases, salivary inhibitors render the virus non-infectious.

Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in water.

The potential for human immunodeficiency virus (HIV) to enter domestic sewers via contaminated body fluids such as blood has spurred interest in the survival of this virus in water and wastewater. This study focused on establishing the inactivation of HIV and productively infected lymphocytes in dechlorinated tap water. In addition, HIV survival was compared with that of poliovirus. Results indicated that either free HIV or cell-associated HIV was rapidly inactivated, with a 90% loss of infectivity within 1 to 2 h at 25 degrees C and a 99.9% loss by 8 h. In comparison, poliovirus showed no loss of infectivity over 24 h. The presence of human serum in tap water slowed the rate of HIV inactivation through 8 h but did not stabilize the virus through 24 h. In addition, blood from stage IV AIDS patients was introduced into tap water, and the recovery of HIV was monitored by using both an infectivity assay and polymerase chain reaction amplification of viral sequences. Virally infected cells were no longer detectable after 5 min in dechlorinated tap water, while little diminution in amplifiable sequences was observed over 2 h. Thus, detection of viral sequences by polymerase chain reaction technology should not be equated with risk of exposure to infectious HIV.

Effect of vitamin E, phosphorus and sorbitol on growth performance and serum and tissue cholesterol concentrations in the pig.

A total of 180 crossbred, weanling pigs were assigned to five dietary treatment groups: 1) a basal corn-soybean meal diet formulated to current NRC recommendations, 2) basal + monosodium phosphate (2 x NRC P recommendations; P), 3) basal + alpha-tocopheryl acetate (220 IU/kg; E), 4) basal + sorbitol (1% of the diet; S) and 5) basal + PES. Dietary treatments were continued until market weight (104 kg). Blood samples were obtained at 3-wk intervals for analysis of serum alpha-tocopherol, P and total cholesterol. Liver and muscle (semimembranosus) samples were obtained at the end of the starter, grower and finisher phases for determination of total cholesterol concentration. The Ca:P imbalance produced by the high-phosphorus diets (P and PES) increased feed intake during the finisher phase. Dietary treatment did not consistently affect total serum cholesterol at any phase of growth. A transient 21.5% (P less than .05) depression of liver cholesterol concentration was observed in the PES-fed pigs at the end of the starter phase but was not apparent at market weight. A similar trend (nonsignificant) was noted for muscle cholesterol concentration. The present study suggests that the PES diet can decrease tissue cholesterol concentration during the nursery phase, but it remains uncertain whether this transient response is a function of age and(or) diet transition at weaning. Further research is necessary to determine whether this response can be translated to the finishing phase, and thereby reduce carcass cholesterol.

Spectrum of biological properties of human immunodeficiency virus (HIV-1) isolates.

In vitro assessment of biological properties of 14 independent isolates of human immunodeficiency virus type 1 (HIV-1) was performed in order to gain insight into the spectrum of behavioral diversity of HIV-1s and to attempt to identify phenotypic traits that may be eventually correlated with in vivo pathogenesis. All of these biologically cloned isolates were found to spread very slowly in most cell cultures, requiring 8-10 weeks for virus to spread from a few infected cells to around 10(5) cells. If viral synergistic activity was also present, as in HTLV-1-infected cells, HIV-1 spread was greatly accelerated. The isolates varied in their cellular tropisms, having as much as 100,000-fold difference in their tropisms for various human CD4-positive cell lines. Several HIV isolates were dual-tropic for both T and promonocytic cells, but some of these isolates did not readily infect U937 promonocytes while readily infecting THP-1 promonocytes. Both the slow spread and extreme tropisms of HIV-1 isolates have practical implications for titering HIVs and for initiating any studies examining the interaction between a given isolate and any given cell. Some isolates did not score readily by reverse transcriptase assays while others did and this did not reflect the amount of infectious virus produced. These findings raise questions about the reliability of HIV quantitation by RT assay. The HIV isolates further varied in their ability to kill and/or fuse cells, whereas some induced cytopathology more efficiently in a given cell line than others, even though the latter appeared to replicate as well. Finally, most isolates killed cells without syncytia formation, demonstrating that cell-to-cell fusion is a minor mechanism of cytopathology. The properties observed for each HIV isolate appeared to be stable phenotypes for that virus and the diversity of biological behavior raises the possibility that independent HIV isolates may differ in their virulence properties in vivo as well.

Expression of the c-rel and c-myc proto-oncogenes in avian tissues.

Reticuloendotheliosis virus (REV-T) transforms very immature avian lymphoid cells and induces a rapidly fatal lymphoma. The viral oncogene, v-rel, encodes a 59 kDa phosphoprotein which is complexed with cellular proteins in the cytosol of REV-T transformed lymphoid cell lines. The proto-oncogene, c-rel, has been highly conserved among both vertebrate and invertebrate species. The expression of both the c-rel and c-myc protoncogenes was characterized during avian development. Two distinct rel-related transcripts were detected. A 4.0kb mRNA was the principal transcript expressed in cells of hematopoietic origin with highest levels detected in bursa, thymus, and spleen tissue obtained from hatched birds. Relatively low levels of this 4.0 kb transcript were detected in nonhematopoietic tissues. Thus, the distribution of this mRNA correlated with the presence of target cells for transformation by v-rel. The 4.0 kb c-rel transcript had a half-life of nearly 2 h in an avian lymphoid cell line. A second rel-related transcript was identified in the ovary of young hens and appeared to be expressed either in primary oocytes or in the developing follicle. This 2.6 kb c-rel mRNA was detected at substantially lower levels in cells of hematopoietic origin. Using radiolabeled RNA probes, the 2.6 kb c-rel transcript was not detected in liver, brain, testes, or muscle. The c-myc proto-oncogene was also expressed in cells of hematopoietic tissue and ova obtained from hatched birds. While intermediate RNA levels were observed in muscle and liver cells, the c-myc gene was not expressed in avian testes.

Expression of the v-rel oncogene in reticuloendotheliosis virus-transformed fibroblasts.

Reticuloendotheliosis virus (REV-T) induces a rapidly fatal lymphoma in chickens through the expression of its oncogene, v-rel, REV-T also morphologically transforms avian fibroblasts in vitro. These transformed cells displayed limited anchorage-independent growth and reached higher saturation density than uninfected or REV-A-infected fibroblasts. Morphologically transformed fibroblasts were tumorigenic when injected into the wing web of chickens. In transformed fibroblasts, the v-rel oncogene was expressed as a 57 kDa phosphoprotein with a half-life of 2 to 4 hr. A cellular phosphoprotein of about 40 kDa was also observed in immunoprecipitates of transformed fibroblasts. The subcellular location of the v-rel-encoded protein was determined using cell fractionation procedures and immunofluorescent staining. In acutely infected, nontransformed fibroblasts, pp57v-rel was associated with the nuclear region, but in morphologically transformed cells the v-rel protein was found in the cytoplasm. These observations suggest that the expression of the v-rel oncogene is insufficient for transformation and that the cellular localization of this transforming protein to the cytoplasm may be required for the progression to an altered cell phenotype in avian fibroblasts.

Transformation of avian lymphoid cells by reticuloendotheliosis virus.

Avian reticuloendotheliosis virus (REV-T) is the most virulent of all retroviruses, inducing an invariably fatal leukemia in chickens with a latent period of 7-10 days. Unlike avian cells transformed by other acutely transforming viruses, lymphoid cells transformed by REV-T are immortalized. Furthermore, in vitro derived, REV-T transformed cells which do not produce virus are tumorigenic and induce lethal reticuloendotheliosis when injected into histocompatible birds. Thus REV-T transforms its target cell both in vitro and in vivo. In addition this transformation is independent of any helper virus functions. Like other acute leukemia viruses, REV-T is replication-defective and must co-replicate with a reticuloendotheliosis associated virus (REV-A). During evolution, a substantial portion of its genome has been deleted and replaced with a host-derived genetic sequence, designated v-rel. Presumably, the v-rel oncogene was transduced from a normal turkey DNA locus, c-rel. There are 9 regions of homology between c-rel and v-rel, however, several differences exist between these genes, suggesting that transformation by REV-T results from the production of an altered v-rel protein. The v-rel sequence is distinct from other known oncogenes and encodes a 57-kDa phosphoprotein. In REV-T transformed cells, this pp57v-rel protein is localized in the cytoplasm. The product of the v-rel oncogene is present at a low level, representing only about 0.003% of total methionine-labelled protein. In addition, pp57v-rel is relatively stable, having an estimated half-life of 4-10 h. The v-rel protein when purified close to homogeneity is complexed with a 40-kDa cellular phosphoprotein in transformed lymphoid cells and possesses serine kinase activity. This review discusses the molecular aspects of transformation by REV-T in the context of other oncogene-encoded proteins.

Round robin investigation of methods for recovering human enteric viruses from sludge.

To select a tentative standard method for detection of viruses in sludge the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group initiated round robin comparative testing of two procedures that, after initial screening of several methodologies, were found to meet the basic criteria considered essential by the task group. Eight task group member laboratories agreed to perform round robin testing of the two candidate methods, namely, The Environmental Protection Agency or low pH-AlCl3 method and the Glass or sonication-extraction method. Five different types of sludge were tested. For each particular type of sludge, a single laboratory was designated to collect the sludge in a single sampling, make samples, and ship it to the participating laboratories. In most cases, participating laboratories completed all the tests within 48 h of sample arrival. To establish the reproducibility of the methods, each laboratory tested each sludge sample in triplicate for the two candidate virus methods. Each processed sludge sample was quantitatively assayed for viruses by the procedures of each individual round robin laboratory. To attain a more uniform standard of comparison, a sample of each processed sample from all laboratories was reassayed with one cell line and passage number by a single laboratory (Environmental Protection Agency Environmental Monitoring and Support Laboratory, Cincinnati, Ohio). When the data were statistically analyzed, the Environmental Protection Agency method was found to yield slightly higher virus recoveries for all sludge types, except the dewatered sludge. The precisions of both methods were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

A report on the AANA Manpower Study--Part I.

Future reports to be released by the Center for Cybernetic Studies will present the occupational structure of the nurse anesthetist and will combine demographic data with a task analysis of the profession. This report has selected some variables of interest and analyzed them. Most important, however, is the intended flexibility that this data based information system should offer. For example, human resource modeling will forecast the number of nurse anesthetists who remain in or leave a given region. This modeling capability could aid schools of anesthesia in their curriculum planning by showing which tasks are predominant in a given location. It could also aid members by providing pertinent employment information. Future reports will present a scenario to exemplify how a data based information system can derive a series of human resource models. The purpose of such an exercise is to develop for the AANA the planning methods some of the larger corporations are already utilizing. The difference, of course, is that these planning methods could be used by the professional association of nurse anesthetists for the advantage of its members. This article has presented a short review of the Manpower Study. We welcome your ideas and suggestions to aid us in making future analyses of this information.

Recovery of indigenous viruses from wastewater sludges, using a bentonite concentration procedure.

A distilled water elution-bentonite concentration technique was developed and used to monitor indigenous viruses present in liquid sludges undergoing land application at six field sites.