Realigning the conventional routes of transmission: an improved model for occupational exposure assessment and infection prevention.

Current recommendations for standard and transmission-based precautions in place for patients who are suspected or known to be infected or colonized with infectious agents are best suited to prevent the transfer of micro-organisms to other patients - that is, to prevent the acquisition of a healthcare-associated infection, rather than to protect the healthcare worker from self-contamination resulting in a potential occupationally acquired infection. This article reviews current recommended infection prevention and control practices and offers a framework for better protection and controls from an occupational health point of view. We offer a model with two exposure routes - contact and aerosol - resulting from work activities and environments, shifting the focus away from particular pathogenic micro-organisms' typical methods for spreading to patients or to other non-workers in hospital and community settings.

Oncolytic virotherapy for ovarian carcinomatosis using a replication-selective vaccinia virus armed with a yeast cytosine deaminase gene.

In this study, we assessed the ability of a highly tumor-selective oncolytic vaccinia virus armed with a yeast cytosine deaminase gene to infect and lyse human and murine ovarian tumors both in vitro and in vivo. The virus vvDD-CD could infect, replicate in and effectively lyse both human and mouse ovarian cancer cells in vitro. In two different treatment schedules involving either murine MOSEC or human A2780 ovarian carcinomatosis models, regional delivery of vvDD-CD selectively targeted tumor cells and ovarian tissue, effectively delaying the development of either tumor or ascites and leading to significant survival advantages. Oncolytic virotherapy using vvDD-CD in combination with the prodrug 5-fluorocytosine conferred an additional long-term survival advantage upon tumor-bearing immunocompetent mice. These findings demonstrate that a tumor-selective oncolytic vaccinia combined with gene-directed enzyme prodrug therapy is a highly effective strategy for treating advanced ovarian cancers in both syngeneic mouse and human xenograft models. Given the biological safety, tumor selectivity and oncolytic potency of this armed oncolytic virus, this dual therapy merits further investigation as a promising new treatment for metastatic ovarian cancer.

Targeted therapy for malignant melanoma.

There has been much development in the field of targeted therapy for melanoma stemming from efforts to decrease treatment-related toxicities and enhance specific cytotoxicity. This review focuses on three modalities of targeted melanoma therapy based on the biology of the targeting mechanism. The first of these modalities is immunotherapy, which functions to generate a specific antimelanoma immunity. A second modality utilizes metabolic pathways of melanin synthesis to target melanoma cells specifically. A third modality ensues from recent advances in molecular biology and the identification of genes responsible for the malignant transformation of normal melanocytes to melanomas. This work has furthered our understanding of the basis of malignancy, as well as the development of novel strategies aimed at targeting aberrant growth in melanoma.

Microcirculation patterns other than loops and networks in choroidal and ciliary body melanomas.

PURPOSE: To provide ophthalmologists and pathologists with expanded criteria for separating patients at high risk of metastatic melanoma from those at low risk on the basis of microcirculation patterns in choroidal and ciliary body melanomas. DESIGN: Tissue culture studies and observational case series. PARTICIPANTS: The pattern-forming ability of four uveal melanoma cell lines of varying degrees of aggressive behavior was studied in vitro. Histologic sections of 234 eyes removed for choroidal or ciliary body melanoma were studied for the presence of microcirculation patterns. METHODS: The study was divided into two phases: the study of histologic sections of eyes removed for choroidal and ciliary body melanomas and observations on the in vitro behavior of cultured melanoma cells of varying degrees of invasive behavior. The presence or absence of each of nine microcirculation patterns was recorded from tissue sections, and interrelationships between different patterns were explored statistically. In vitro reconstitution of patterns and a study of the interrelationships of patterns in histologic sections was carried out. In the in vitro studies, uveal melanoma cell lines of varying degrees of aggressive potential were cultured to observe the development of architectural patterns other than loops and networks. MAIN OUTCOME MEASURES: In histologic studies, the outcome measure was the conditional probability of detecting loops or networks given the presence or absence of other patterns positive for periodic acid-SCHIFF: For tissue culture studies, the outcome measure was either the development or lack of development of patterns of different shapes in vitro. RESULTS: Histologic studies disclosed that given the presence of arcs without or with branching in a tissue section, it is likely that loops or networks will be detected in the same section plane, suggesting that the production of these patterns by aggressive tumor cells reflects a spectrum of architectural potential. In vitro studies confirmed this hypothesis by revealing that highly aggressive and metastatic uveal melanoma cell lines, but not poorly aggressive tumor cell lines, generated parallel channels with and without crosslinking and arcs with and without branching as well as loops and networks. CONCLUSIONS: The criteria for separating patients into low- and high-risk categories for metastasis from uveal melanoma should be expanded to include patterns other than loops or networks. In both the pathology laboratory as well as in a clinical setting, the detection of arcs or arcs with branching and parallel channels should prompt a careful search for loops and networks and for crosslinking parallel channels, respectively.

A novel approach for the identification of unique tumor vasculature binding peptides using an E. coli peptide display library.

BACKGROUND: Tumor neovascularization is necessary for continued tumor growth and metastasis. During the process of endothelial cell (EC) recruitment and tumor infiltration, specific molecular markers unique for this interaction are expressed on the EC surface. Targeting these molecular markers would, in effect, allow for specific tumor targeting. Tripeptide sequence motifs have previously been reported that will bind to angiogenic tumor ECs. These sequences were identified from in vivo phage peptide display libraries. The purpose of this study was to use a more simplified bacterial peptide display library in an in vitro system to seek out peptide motifs with unique binding to tumor microvasculature. METHODS: FliTrx is a bacterial peptide display library containing the entire repertoire of possible random dodecapeptides expressed on the flagella tip of E. coli. Two EC populations were used for the screening process, Matrigel invading cells (MAGIC) and tumor-derived endothelial cells (TDEC). MAGIC are obtained from ECs that infiltrate a subcutaneous fibroblast growth factor-containing Matrigel deposit, and TDEC are ECs selectively obtained from tumor vasculature. FliTrx cells were incubated with MAGIC at 4 degrees C to remove any potential clones displaying peptides that will bind to nonspecific EC surface targets. The non-binding cells were then incubated with TDEC, allowing for clones displaying potential binding peptides to bind tumor specific targets on TDECs. The bacterial population was then expanded and this "panning" process was carried out a total of five times. Peptide insert sequences from 100 bacterial colonies were analyzed for potential repetitive peptide motifs. RESULTS: Recurring peptide sequences were detected that were 3-mers (13 sequences) and 4-mers (4 sequences). Of the 3-mers, four repeated 3 times, whereas none of the 4-mers repeated more than twice. All of the repeated sequences were basic in charge, and arginine was the most commonly seen amino acid. A tripeptide basic-basic-nonpolar amino acid arrangement was the most prevalent charge sequence in all repetitive motifs (17 repeat sequences). Two test peptides showed TDEC binding specificity, and both conformed to the basic-basic-nonpolar motif. CONCLUSIONS: We report peptide sequences derived from panning an in vitro system designed to detect tumor-EC specific markers. These putative motifs may serve as molecular determinants for a novel therapeutic modality aimed at specifically targeting tumors through tumor angiogenic vessels.

Auditory evoked responses and learning and awareness during general anesthesia.

BACKGROUND: There is a major distinction between conscious and unconscious learning. Monitoring the mid-latency auditory evoked responses (AER) has been proposed as a measure to ascertain the adequacy of the hypnotic state during surgery. In the present study, we investigated the presence of explicit and implicit memories after anesthesia and examined the relationships of such memories to the AER. METHODS: We studied 180 patients scheduled for elective surgical procedures. After a thiopental induction, one of four anesthetics were studied: Opioid bolus: 7.5 microg x kg(-1) fentanyl, 70% N2O, with 2.5 microg x kg(-1) supplements as needed (n=100); Opioid infusion: Alfentanil 50 microg x kg(-1) bolus, 1-1.5 microg x kg(-1) x min(-1) infusion, 70% N2O (n=40); Isoflurane 0.3%: Fentanyl 1 microg x kg(-1), 70% N2O, isoflurane 0.3% expired (n=16); Isoflurane 0.7%: Fentanyl 1 microg x kg(-1), 70% N2O, isoflurane 0.7% expired (n=23). AER were recorded before anesthesia, 5 min after surgical incision and then every 30 min until the end of surgery. A tape of either the story of the "Three Little Pigs" or the "Wizard of Oz" was played continuously between the recordings. Explicit memory was assessed postoperatively by tests of recall and recognition, and implicit memory was assessed by the frequency of story-related free associations to target words from the stories, which were solicited twice during a structured interview. RESULTS: Six patients showed explicit recall of intraoperative events: All received the opioid bolus regimen. About 7% of patients reported dreaming during anesthesia. The incidence of picking the correct story that had been presented during anesthesia averaged 49%, i.e., very close to chance level. Overall, priming occurred only at the second association tests for the opioid bolus regimen, for which the frequency of an association to the presented story among those not giving an association to the control story was 26%, which was double the frequency (13%) of an association to the control story among those not giving an association to the presented story. This was significant by McNemar's test, P=0.02. There were significant associations between awareness, priming and AER, e.g., recall was associated with higher Nb amplitudes during anesthesia and priming was associated with shorter wave latencies. CONCLUSIONS: The incidence of awareness in patients anesthetized with nitrous oxide and bolus supplementation was 6%. Thus, this anesthetic technique did not reduce the risk of awareness compared with the use of nitrous oxide alone. Implicit memory occurred during nitrous oxide and bolus supplementation. Recording AER during anesthesia may help to predict awareness and implicit memory, particularly the former. The short contents of most of the dreams which were recalled could hamper future studies in this area.

Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant.

Tumor-directed gene therapy, such as "suicide gene" therapy, requires high levels of gene expression in a high percentage of tumor cells in vivo to be effective. Current vector strategies have been ineffective in achieving these goals. This report introduces the attenuated (thymidine kinase (TK)-negative) replication-competent vaccinia virus (VV) as a potential vector for tumor-directed gene therapy by studying the biodistribution of VV in animal tumor models. A TK-deleted recombinant VV (Western Reserve strain) expressing luciferase on a synthetic promoter was constructed. Luciferase activity was measured in vitro after transduction of a variety of human and murine tumor cell lines and in vivo after intraperitoneal (i.p.) delivery in C57BL/6 mice with 7-day i.p. tumors (10(6) MC-38 cells). Three other in vivo tumor models were examined for tumor-specific gene expression after intravenous delivery of VV (human melanoma in nude mice, adenocarcinoma liver metastasis in immunocompetent mice, and subcutaneous sarcoma in the rat). In addition, a replication-incompetent vaccinia (1 microg of psoralen and ultraviolet light, 365 nm, 4 minutes) was tested in vitro and in vivo and compared with active virus. Luciferase activity in i.p. tumors at 4 days after i.p. injection of VV was >7000-fold higher than lung, >3000-fold higher than liver, and >250-fold higher than ovary. In addition, intravenous injection of VV resulted in markedly higher tumor luciferase activity compared with any other organ in every model tested (up to 188,000-fold higher than liver and 77,000-fold higher than lung). Inactivation of the virus resulted in negligible gene expression in vivo. In summary, VV has a high transduction efficiency in tumor cells with high levels of gene expression. The results suggest a selective in vivo replication of TK-deleted VV in tumor cells. Replication competent, TK-deleted VV appears to be an ideal vector for testing the in vivo delivery of toxic genes to tumor cells.

Late effects in children treated with radiation therapy for Wilms' tumor.

PURPOSE: To determine the frequency and types of late effects in children receiving radiation therapy (RT) for Wilms' tumor. MATERIALS AND METHODS: From 1968 to 1994, 55 children received megavoltage RT at our institution as part of treatment for Wilms' tumor. A total of 42 (76.4%) have survived and have a minimum follow-up of 5 years. There were 25 female and 17 male patients with a median age at diagnosis of 48 months (range, 7-126 months). There were 12 Stage I, eight Stage II, 15 Stage III, six Stage IV, and one Stage V patient. RT was delivered to the hemiabdomen in 36 and whole abdomen in six patients. RT dose was 1000-1200 cGy (Group A) in 12, 1201-2399 cGy (Group B) in 11, and 2400-4000 cGy (Group C) in 19. Whole-lung RT was delivered to 13 patients either at diagnosis or pulmonary relapse. All patients received chemotherapy; the most common agents were actinomycin-D/vincristine/adriamycin in 13 and actinomycin-D/vincristine in 18. Median follow-up was 181 months (range, 60-306 months). RESULTS: Of 42 patients, 13 (31.0%) did not have late effects of treatment. The number of patients who developed muscular hypoplasia, limb length inequality, kyphosis, and iliac wing hypoplasia were seven (16.7%), five (11.9%), three (7.1%), and three (7.1%), respectively. Scoliosis was seen in 18 (42.9%) with only one patient requiring orthopedic intervention. Median time to development of scoliosis was 102 months, with a range of 16-146 months. The actuarial incidence of scoliosis at 5, 10, and 15 years after RT was 4.8 +/- 3.3%, 51.8 +/- 9.0%, and 56.7 +/- 9.3%, respectively. Only one of 12 Group A patients developed scoliosis. The 10- and 15-year actuarial incidences of scoliosis for Group A and B patients were 37.7 +/- 12.4% and 37.7 +/- 12.4%, whereas for Group C patients the incidences were 65.8 +/- 12.0% and 74.4 +/- 11. 7% (p = 0.03, log rank test). The actuarial incidence of bowel obstruction at 5, 10, and 15 years was 9.5 +/- 4.5%, 13.0 +/- 5.6%, and 17.0 +/- 6.5%. Of 23 patients, five irradiated within 10 days of surgery and one of 19 irradiated after 10 days developed bowel obstruction (p = 0.09, log rank test). Three patients developed hypertension with normal blood urea nitrogen (BUN) and creatinine levels; another patient had chronic renal insufficiency in a nonirradiated kidney. One patient developed diffuse interstitial pneumonitis. Of the 19 female patients who have reached puberty, three have given birth, and 15 have regular and one has irregular menstrual periods. Four patients developed benign neoplasms; three were in the RT field (two osteochondroma, one lipoma) and one outside (cervical intraepithelial neoplasia II). There were three second malignancies (chronic myelogenous leukemia at 9 years, osteosarcoma at 11 years, and breast cancer at 25 years after initial diagnosis of nephroblastoma); both solid malignancies occurred in the RT field. CONCLUSIONS: Late effects of therapy were seen in more than two thirds of children treated for Wilms' tumor. Children treated with lower doses (<2400 cGy) had a lower incidence of scoliosis compared with those who received more than 2400 cGy. There is also a suggestion that the incidence is lower in patients who received 1000-1200 cGy. Severe physical and functional deformity from RT was uncommon.

Augmentation of melanoma-specific gene expression using a tandem melanocyte-specific enhancer results in increased cytotoxicity of the purine nucleoside phosphorylase gene in melanoma.

The lineage-specific human tyrosinase promoter has been used to successfully target gene expression at the transcriptional level to melanoma cells. The tyrosinase promoter, alone and in combination with a single, or a dual, tandem melanocyte-specific enhancer, was used to regulate expression of the firefly luciferase reporter gene. Transient transfections of these tissue-specific luciferase constructs in human and murine melanoma (Pmel, B16mel) and colon carcinoma (WiDr, MC38) cell lines resulted in melanoma-specific luciferase expression that was amplified 5- and 500-fold with the addition of a single or double enhancer, respectively, to the tyrosinase promoter. When the double enhancer-promoter construct expressed the highly toxic Escherichia coli purine nucleoside phosphorylase (PNP) gene, transfection of the same cell lines followed by administration of the prodrug 6-methyl purine deoxyriboside (6-MPDR) at a concentration of 50 microM caused melanoma-specific in vitro cell killing. Within 5 days after prodrug administration methylthiazol-tetrazolium (MTT) cytotoxicity assays showed that only 15 and 9% of Pmel and B16mel cells, respectively, remained viable compared with controls. This effect was highly specific, as 90 and 96% of WiDr and MC38 colon carcinoma cells remained viable 5 days after identical treatment. This effect was a direct result of increased tissue-specific conversion of 6-MPDR to the toxic metabolite 6-methylpurine (6-MP), as documented by HPLC analysis of culture supernatants. These results show that the dual tandem melanocyte-specific enhancer provides powerful amplification of the transcriptional targeting of gene expression afforded by use of the tyrosinase promoter. This amplification translates into increased, highly specific cytotoxicity to melanoma by the PNP/6-MPDR enzyme/prodrug system and, therefore, has potential efficacy in the use of gene therapy for the treatment of metastatic melanoma.

Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy.

Tumor-directed gene therapy faces many obstacles. Lack of tissue targeting and low in vivo transduction efficiency represent some of the limitations for a successful therapeutic outcome. A thymidine kinase-deleted mutant vaccinia virus has been shown in marker studies to replicate selectively in tumor tissue in animal models. Purine nucleoside phosphorylase (PNP), from E. coli, converts the nontoxic prodrug 6-methylpurine deoxyriboside (6-MPDR) to the toxic purine 6-methylpurine. In this study, we investigated the cytotoxic properties of PNP, expressed by an optimized synthetic early/late promoter in a vaccinia virus (vMPPNP). In vitro cytotoxicity of psoralen-inactivated vMPPNP (1 microg of psoralen, 4 min of LWUV [365 nm]) at the maximum tolerated dose (MTD) of 6-MPDR (80 microM) reduced cell viability by day 3 to 1.7%. At an MOI of 0.002, replication-competent vMPPNP and 6-MPDR (80 microM) caused reduction of cell viability to 19.8% within 4 days. Furthermore, there was complete abrogation of viral replication after intracellular conversion of prodrug into the active toxin. The potency of such a system was similar among all histologies tested. Finally, the cytotoxic efficacy has been shown to be more rapid and complete than that of cytosine deaminase (CD), a more established enzyme/prodrug system. When virus was delivered intraperitoneally into athymic mice with hepatic metastases, followed by administration of prodrug, there was a significant prolongation of survival and a 30% cure rate. In summary, owing to its tumor-targeting capabilities, high transduction efficiency, and high gene expression, a vaccinia virus expressing PNP could prove to be a potent and valuable vector for tumor-targeted gene therapy.

Augmented capillary leak during isolated hepatic perfusion (IHP) occurs via tumor necrosis factor-independent mechanisms.

Isolated organ perfusion of the liver or extremity with tumor necrosis factor (TNF) and melphalan results in regression of bulky tumors in the majority of patients. The efficacy of TNF in this setting is not known, although data suggest that it may exert antitumor effects primarily on tumor-associated neovasculature. We studied the effects of TNF on capillary leak in liver and tumor tissue during isolated hepatic perfusion (IHP) with melphalan. Twenty-seven patients with unresectable cancer confined to the liver underwent a 60-min hyperthermic IHP using 1.5 mg/kg melphalan alone (n = 7) or with 1.0 mg of TNF (n = 20). Complete vascular isolation was confirmed in all patients using an intraoperative leak monitoring I-131 radiolabeled albumin technique. Samples of tumor and liver were collected just prior to and immediately after IHP. There was no difference in I-131 radiolabeled cpm/g of tissue (cpm) in liver versus tumor at baseline (P2 = 0.44). After IHP, I-131 albumin cpm were higher in tumor versus liver (10,999 +/- 1,976 versus 3,821 +/- 780, respectively; P2 < 0.005). However, I-131 albumin cpm in tumor were not effected by TNF (11,636 +/- 2,518 with TNF versus 9,180 +/- 2,674 without TNF; P2 = 0.59). TNF did not affect melphalan concentrations in tumor (1,883 +/- 540 ng/g versus 1,854 +/- 861 ng/g without TNF; P2 = 0.9). Capillary leak, as reflected by diffusion of I-131 radiolabeled albumin into the interstitial space, is comparable in liver and tumor before IHP but is significantly higher in tumor after IHP. The increased diffusion in the capillary tumor bed must occur through TNF-independent mechanisms such as intrinsic features of tumor neovasculature, hyperthermia, or other unrecognized perfusion-related factors. These data indicate that TNF must continue to be critically evaluated in clinical trials before it is routinely used with melphalan in isolated organ perfusion.

A model of the quaternary structure of enolases, based on structural and evolutionary analysis of the octameric enolase from Bacillus subtilis.

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pHo) for activity is 8.1-8.2, and the Km for 2-PGA is approximately 0.67 mM. Using the dimeric Calpha structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86-96) and the loop between helix L and strand 1 (HL-S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL-S1 loop is truncated by 4-6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL-S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.

Intraarterial calcium stimulation and intraoperative ultrasonography in the localization and resection of insulinomas.

BACKGROUND: Standard imaging studies (computed tomography, magnetic resonance imaging, somatostatin receptor scintigraphy, ultrasonography, and angiography) correctly localize insulinomas in less than 50% of patients and provide no information about the feasibility of enucleation based on proximity of tumor to pancreatic duct. We reviewed our experience with intraarterial calcium stimulation (Ca-Stim) and intraoperative ultrasonography (IOUS) to localize and guide management of insulinomas. METHODS: Thirty-six patients (14 men, 22 women, median age 44 years) with insulinomas were treated between August 1989 and June 1996. Preoperative imaging studies were obtained. Patients underwent abdominal exploration with IOUS. Fourteen were evaluated by a surgeon blinded to preoperative imaging results. RESULTS: Tumors (4 to 50 mm) were resected by enucleation (67%) or partial pancreatectomy (33%); all were cured. Sensitivities of computed tomography, magnetic resonance imaging, somatostatin receptor scintigraphy, ultrasonography, angiography, and Ca-Stim in localizing insulinomas were 24%, 45%, 17%, 13%, 43%, and 94%, respectively. Tumors were identified by blinded surgical exploration with IOUS in 12 of 14 patients (86%). CONCLUSIONS: All insulinomas were identified before operation; however sensitivity of individual noninvasive tests was low (less than 50%). In contrast, Ca-Stim was correct in 94% of cases, thus allowing a focused pancreatic exploration and obviating use of blind distal pancreatectomy. IOUS can then be used to guide safe enucleation.

Survival of patients with localized prostate cancer treated with percutaneous transperineal placement of radioactive gold seeds: stages A2, B, and C.

Between 1984 and 1991, a total of 177 patients with adenocarcinoma of the prostate were treated with transcutaneous, transperineal radioactive gold seeds. Of these 177 patients, 20 were determined to have pelvic lymph node involvement and were excluded from this review. The remaining 157 patients received a median radioactivity dose of 164 mCi with a median follow-up of 48 months. Cancer-specific survival at 5 years was 100% for stage A2 and B1, 90% for stage B2, and 76% for stage C cancer. Covariates of grade, total radioactivity administered, age of the patient, and number of seeds implanted did not influence disease-free survival in a statistically significant manner. Significant complications were observed in two patients. The survival rates of patients treated with 198Au seed implantation for localized cancer are equivalent or better when compared to historical data of patients treated with 125I implantation, external beam radiotherapy, combination radioactive gold seed implantation and external irradiation, and radical prostatectomy. In addition, these comparable survival rates using interstitial 198Au seeds may be achieved with less morbidity.

Long-term speech results of cleft palate patients with primary palatoplasty.

This investigation examined the influence of cleft type, type of surgery, age at surgery, and gender on speech proficiency of 204 patients with cleft palate who required only primary palatoplasty. Speech measures were obtained for each subject from at least three annual examinations between the ages of 4 and 16 years. Neither age at surgery nor type of surgery were discriminating factors. The less extensive cleft type, i.e., soft palate only, was associated with greater rates of change in the performance variables than were the other three cleft types. Females showed greater rate changes than males.

Intraosseous transfusion in an anesthetized swine model using 51Cr-labeled autologous red blood cells.

Peripheral venous access can often be difficult to obtain in infants and young children. Landmark articles in the 1940s showed that the intraosseous (IO) route was a viable one for resuscitation. While anecdotal reports and clinical experience suggest that blood products can be transfused via the IO route, it has not been specifically studied nor documented. We performed a prospective study to document the feasibility of red blood cell transfusion via the IO space. We studied the rapid infusion of 51Cr-labeled red blood cells via the IO space through an 18-gauge IO needle in three normovolemic immature swine. Serial central venous samples were removed at 30 seconds and at 1, 5, 15, 30, and 60 minutes and analyzed for evidence of radiolabeling. Our results revealed rapid delivery of radiolabeled red blood cells into the central circulation with no evidence of early heomolysis. Highest counts were seen in samples taken at 30 seconds to 1 minute. We conclude that the IO route is a viable means for blood transfusion in a nonhemorrhagic model.

The nature of naturally occurring mutations in the hemagglutinin gene of vaccinia virus and the sequence of immediately adjacent genes.

The expression of the vaccinia IHD-J hemagglutinin (HA) gene is regulated by two promoters, an early/late and a second distinct late promoter. The first promoter results in transcripts that begin early and are synthesized throughout the infection. All transcripts from this promoter initiate at the same 5' site. A second promoter is only active late in infection and initiates transcription some distance upstream of the first promoter. We have previously shown that the transcriptional start site controlled by this second, "late only" promoter lies within the coding sequence of an upstream reading frame (p16-ORF), whereas the termination of early transcription of the HA gene utilizes a transcription termination signal (TTTTTNT) located just beyond the coding region of an immediately downstream reading frame (p17-ORF). In order to assist our understanding of HA gene expression, we report here the sequence of these two ORFs adjacent to the HA gene. The HA-ORF itself consists of 945 bp, whereas the upstream p16-ORF consists of 429 bp and the downstream p17-ORF of 453 bp, sufficient to encode polypeptides of 16 and 17 kD, respectively. While many strains of vaccinia are HA+, rabbitpox virus and the variant of vaccinia IHD-J, designated IHD-W, are HA-. We report and compare here the HA gene sequences of wild-type rabbit poxvirus, two spontaneous HA+ revertants of rabbit poxvirus, and the HA- vaccinia strain IHD-W to that of the previously sequenced (1) prototype HA+ IHD-J strain of vaccinia. All differences were found to occur within the HA open reading frame.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of the vaccinia virus hemagglutinin gene.

The vaccinia virus hemagglutinin (HA) is a glycoprotein found on the plasma membrane of infected cells and the envelope of extracellular virus. Two forms of HA (85 and 68 kDa) are detected by immunoblot analysis. Although hemagglutination activity is only readily detectable late in infection, the 85-kDa HA appears early and accumulates throughout infection, whereas the 68-kDa form appears only late in the cycle. Production of the 68-kDa HA but not the 85-kDa HA was inhibited by either cytosine arabinoside or rifampin. Analysis of HA gene expression reveals a complex pattern of expression. The HA gene is transcribed early to yield a 1.65-kb dicistronic early transcript, consisting of the 945-bp HA open reading frame (ORF) fused to a 453-bp downstream ORF. Transcription from this site initiates 7 bases upstream of the AUG initiating codon of the HA ORF. Due to the discrepancy between the calculated size of the HA protein (33 kDa) and that reported for the unglycosylated HA protein derived from in vitro translation (58 kDa), we placed an early transcription termination signal (TTTTTAT) directly downstream of the 945-bp HA ORF. This led to a reduction in size of the early HA mRNA to 1.2 kb, as expected, but had no effect on the formation of either the 85- or 68-kDa protein. Transcripts originating from the early promoter are found throughout the infection cycle. However, after DNA replication, transcription from a second, late promoter ensues. The transcriptional start site of the late promoter is within a consensus TAAATG sequence located 135 bases upstream of the transcriptional start site of the first promoter. The late transcriptional start site is also found within an upstream ORF.

Velar-pharyngeal status in cleft palate patients with expected adenoidal involution.

This study was designed to provide information about whether cleft palate patients with hypertrophied adenoids maintain velar-pharyngeal contact during the time of expected adenoidal atrophy. Thirty-nine subjects were selected from a large longitudinal study on the basis of availability of lateral still x-ray films taken in series from 5 to 16 years of age. Ratings of velar-pharyngeal contact and ratings of adenoid size were obtained from the films. The obtained data indicated the expected decrease in adenoid size but also, for the group, maintenance of velar-pharyngeal contact. However, three of the 39 subjects were judged to show loss of such contact during the period of study, and an additional four had surgery for velopharyngeal incompetence after the completion of the study. All seven appeared to show significant deterioration of velopharyngeal status in middle or late adolescence. Implications of these findings are discussed.