An improved vector for high-level, consistent retroviral transgene expression in human thymocytes after competitive reconstitution from transduced peripheral blood stem cells.

One problem in hematopoietic stem cell (HSC)-based gene therapy is the low-level, and often transient, transgene expression in progeny cells in vivo. Here we have evaluated retroviral vector designs for improved long-term in vivo transgene expression levels in thymocytes recovered after transplantation of gene-modified HSCs. First, several vector designs were screened in vitro by single-cell analysis of transgene marking and expression to rapidly identify optimal vectors for sensitive tracking of marked cells. Next, using one optimal vector, we show that gene-modified HSCs can competitively reconstitute thymopoiesis in SCID-hu thymus/liver mice, with transgene expression detectable on 0-40% of marked donor thymocytes. Modified vector designs (termed MSCV-SAR and MoMLV-SAR), which enhance transgene expression in primary T cells in vitro, were shown here to improve in vivo transgene expression levels per cell 12- to 14-fold (mean fluorescence intensity was 2175 for MSCV-SAR vs. 174 for LNGFRSN; %NGFR(+) donor(+) cells with high-level expression was 58% for MSCV-SAR vs. 4% for LNGFRSN). Importantly, 61% of grafts had high-level transgene expression on thymocytes with the MSCV-SAR vector versus 0% of grafts for LNGFRSN or MoMLV-SAR. Transgene expression was demonstrated in various stages of thymocyte differentiation and was consistently detected in early thymic progenitors. We suggest that the MSCV-SAR vector described here is particularly advantageous for applications requiring high-level, consistent transgene expression in a diverse repertoire of T cells derived from gene-modified HSC grafts.

Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells.

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR(+) cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin(-/lo)Sca-1(+) and c-kit(+)Sca-1(+) subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR(+)c-kit(+)Sca-1(+) bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR(+) cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

Hematologic recovery in mice transplanted with bone marrow stem cells expressing anti-human immunodeficiency virus genes.

We have used a mouse bone marrow transplantation (BMT) model to study the safety of retrovirus-mediated transfer of anti-HIV genes (RevM10 and HIV-1 pol antisense) into hematopoietic stem/progenitor cells (HSPCs). In particular, we have monitored the hematologic recovery post-BMT and transgene expression in myeloid and lymphoid lineages, and analyzed tissue sections for evidence of any transgene-related pathological condition. Expression of anti-HIV genes had no effect on kinetics of hematologic recovery post-BMT. The average time to reach 20% of normal cell counts was 15-17 days for white blood cells and 12-14 days for platelets, and the average time to reach complete recovery was 42-56 days for leukocytes and 104-161 days for platelets. Hematocrit levels were not significantly affected by irradiation and transplantation procedures. Donor chimerism was uniformly > or =90% in all transplanted animals. At 4-5 weeks post-BMT transgene expression was detected in peripheral blood leukocytes in 100% of the animals and ranged from 4.5 to 44.7%. In a majority of the animals the percentage of transgene-expressing cells in circulation decreased over time but remained detectable for the length of the study (>6 months). Expression was detected in all analyzed cell lineages (RBCs, platelets, monocytes, granulocytes, and T and B cells). Relative counts of various leukocytes (Mac1+ monocytes, Gr1+ granulocytes, CD3+ T cells, and B220+ B cells) were normal. There were no treatment-related histopathological changes in a wide range of tissues examined. In addition, there were no treatment effects on differential leukocyte counts, and morphology of peripheral blood and bone marrow brush smears. In summary, transfer and expression of the RevM10 and the HIV-1 antisense genes into hematopoietic stem/progenitor cells in vivo appears safe. We propose that the mouse bone marrow transplantation model could be used to evaluate some safety aspects of HSPC-based gene therapies.

Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells.

We have studied retroviral transgene expression in primary human lymphocytes. Our data demonstrate that transgene expression is high in activated primary CD4+ T cells but significantly decreased in mitotically quiescent cells. Incorporation of a DNA fragment from the scaffold attachment region (SAR) of the human beta interferon gene into the vector improved transgene expression, particularly in quiescent cells. The SAR element functioned in an orientation-dependent manner and enhanced expression of Moloney murine leukemia virus- and murine embryonic stem cell-based vectors. Clonal analysis of transduced T cells showed that the SAR sequence did not confer position-independent expression on a transgene but rather prevented the decrease of expression when cells became quiescent. The SAR sequence also enhanced transgene expression in T cells generated from retrovirally transduced CD34-enriched hematopoietic progenitor-stem cells in a SCID-hu thymus-liver mouse model. We have used the SAR-containing retroviral vector to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) Rev gene. Compared to a standard retroviral vector, the SAR-containing vector was up to 2 orders of magnitude more efficient in inhibiting replication of the HIV-1 virus in infected CD4+ peripheral blood lymphocyte populations in vitro. This is the first demonstration that SAR elements can be used to improve retroviral vector expression in human primary T cells.

A role for the Wnt gene family in hematopoiesis: expansion of multilineage progenitor cells.

The microenvironment is a key regulator of hematopoietic stem cells (HSCs) and is a likely source of extracellular factors that control stem cell fate. A better understanding of these microenvironmental factors may come from investigations of developmental cell fate determination in which the critical roles of cell-cell interactions of multipotential cells have been shown. The Wnt gene family is known to regulate the cell fate and cell-cell interactions of multipotential cells in a variety of tissues. Expression of Wnts and of their putative receptors encoded by murine homologs of the Drosophila frizzled gene in hematopoietic tissues was examined by reverse transcriptase-polymerase chain reaction. Wnt-5a and Wnt-10b were expressed in day-11 murine yolk sac, day-14 fetal liver, and fetal liver AA4+ cells. The expression profiles of four murine frizzled homologs, Mfz3-7, were nearly identical to that of Wnt-5a and Wnt-10b. Notably, Wnt-10b was expressed in the fetal liver AA4+ Sca+ c-kit+ flASK) HSC population. A role for Wnts in HSC fate determination was studied by treatment of HSC populations in culture with soluble WNT proteins. The addition of conditioned media from cells transfected with Wnt-1, Wnt-5a, or Wnt-10b cDNAs to cultures of flASK cells stimulated a sevenfold, eightfold, and 11-fold expansion in cell number, respectively, relative to control media. Removal of WNT-5a from this media by immunodepletion depleted the stimulatory activity from the media, whereas addition of a partially purified WNT-5a stimulated a fivefold expansion relative to control cells. Transduction of flASK cells with a retrovirus bearing a Wnt-5a cDNA enhanced proliferation. We conclude that WNTs stimulate the survival/proliferation of hematopoietic progenitors, demonstrating that WNTs comprise a novel class of hematopoietic cell regulators.

Polymerase chain reaction-and RNA hybridization-based method for the investigation of deep-seated candidiasis.

OBJECTIVE: To determine the usefulness of a polymerase chain reaction (PCR) and RNA hybridization method for the diagnosis of invasive candidiasis and to compare its sensitivity with blood cultures. DESIGN: Blood cultures and a blood sample for PCR were taken from patients with suspected invasive candidiasis. A 105 base pair conserved segment within the rDNA of Candida species was amplified. The amplicon was detected by hybridization and gel electrophoresis. SETTING: Intensive care units of two tertiary care hospitals. PATIENTS: One hundred and eighteen patients 16 years of age or older with four more risk factors for invasive candidiasis were enrolled. Present or recent past treatment with broad spectrum antibiotics, cancer chemotherapy, immunosuppressive drugs, granulocytopenia or granulocytosis, intravascular catheterization, tracheal intubation, recent abdominal surgery and parenteral nutrition were considered risk factors. RESULTS: Forty-three patients had invasive candidiasis. PCR detected infections in 28 and 26 patients (sensitivity 65.1% and 60.4%) by hybridization and gel electrophoresis, respectively. The sensitivity of blood cultures was 58.1%. Of 25 patients with positive blood cultures, 17 were positive by PCR with the hybridization method. Eleven patients with invasive candidiasis had negative blood cultures but were positive by PCR. CONCLUSION: PCR, especially with a hybridization detection method, is more sensitive than blood culture for invasive candidiasis and may facilitate the diagnosis of nonfungemic disease.

Standardized gentamicin dosing enhances peak serum concentrations.

The objectives of this study were to determine if a standardized gentamicin dosing protocol would improve clinical effectiveness, yield higher peak serum concentrations, and improve the success rate of attaining peaks in the desired range when compared with empiric dosing practices used by prescribers. The study was conducted as a before-after program effectiveness evaluation in non-critically ill patients, aged 16-65 y with stable renal function, who were prescribed gentamicin. A standardized dose of 2 mg/kg (ideal or adjusted weight) was administered intravenously every 12 h in the intervention phase. Response to therapy (time to defervescence, white cell count, reinstitution of antibiotic therapy), serum concentrations (peaks > 10 mumol/L (5.6 mg/L) and troughs < 4 mumol/L (2.2 mg/L)), and toxicity were monitored in both groups. Thirty-four consecutive patients were enrolled into the control phase and an equal number into the intervention phase. Surgical patients comprised the majority of the study population. Desired peak concentrations were attained in 97% of intervention vs. 59% of control patients (p < 0.001). Mean peak serum concentrations were higher in the intervention phase than in the control phase, 16.1 mumol/L vs. 11.2 mumol/L (p < 0.001), respectively. Median time to become afebrile trended toward a statistical decrease in the intervention as compared to the control group, 3 vs. 5 d (p = 0.076), respectively. There was no significant difference in clinical effectiveness nor in the occurrence of nephro- or ototoxicity. Continued evaluation of this dosing protocol is warranted.

Pseudomonas aeruginosa blepharitis in a patient with vancomycin induced neutropenia.

A patient who developed a necrotizing pseudomonas blepharitis as a complication of drug induced neutropenia is reported. Although the patient's neutrophil count recovered and he survived his infection, radical reconstructive surgery of his eyelids was required. Clinicians should keep in mind that in patients with predisposing risk factors, even commonly encountered infections such as blepharoconjunctivitis may be caused by atypical pathogens.

Short-course itraconazole in the treatment of candida vulvovaginitis: A multicentre Canadian study.

OBJECTIVE: To determine the clinical and mycological effectiveness of oral itraconazole in the treatment of acute candida vulvovaginitis. DESIGN: A prospective, randomized and single-blinded, multicentre trial of 221 women, comparing a one-day course of oral itraconazole 200 mg bid with vaginal clotrimazole 500 mg single-dose therapy. MAIN OUTCOME MEASURES: Symptoms, signs and mycological results were assessed up to two months following treatment. Adverse events were recorded and evidence of hepatotoxicity sought. RESULTS: At 10 and 30 days post-treatment, clinical and mycological cure rates were similar (61.3% clinical and 88.6% mycological 10 days after, and 67.7% clinical and 79.5 mycological 30 days after itraconazole; 64.0 clinical and 85.9% mycological 10 days after, and 62.1% clinical and 78.6 mycological 30 days after clotrimazole) with the majority of both treatment groups free from infection. A total of 69 patients reported adverse events, which were generally transient and mild. Itraconazole was more often associated with gastrointestinal or central nervous system complaints, while clotrimazole recipients more often had genitourinary symptoms. No evidence of hepatotoxicity was found. A higher incidence of relapse was noted among women on the birth control pill and among those who were symptomatic for longer than 10 days before treatment. CONCLUSIONS: A one-day course of oral itraconazole is as effective as intravaginal clotrimazole in the treatment of acute yeast vulvovaginitis. The number of patients reporting adverse events was similar for the treatment groups, although the side effect profile differed. No hepatotoxicity was observed.

Conformity with guidelines for antimicrobial prophylaxis against bacterial endocarditis.

The extent to which prescribed antimicrobial prophylaxis against bacterial endocarditis conformed with American Heart Association (AHA) guidelines was determined and the frequency of nonconformity with specific elements of the guidelines was evaluated. Patients with conditions defined by AHA as placing them at risk for developing endocarditis were identified through medical records for a four-year period at an 850-bed hospital. Data about the procedures they underwent and prophylaxis prescribed were compared with the AHA guidelines. Conformity with the guidelines was evaluated according to whether prophylaxis was recommended, optional, or unnecessary; nonconformity with specific elements of the guidelines (indication, choice of antimicrobial, dose, dosage interval, timing, and duration) was also evaluated. The following variables were evaluated for possible association with nonconformity to the guidelines: patient's age and sex, penicillin allergy, use of a consultant, and whether the procedure was the first performed in the patient after identification of the cardiac condition. Of the 131 cases analyzed, 29 (22%) involved prophylaxis that conformed with the AHA guidelines. Conformity with the guidelines was significantly lower when prophylaxis was recommended or optional than when it was unnecessary. Nonconformity was most common with the following elements: indication, choice of antimicrobial, and dose. Recommended prophylaxis was given more often in children than in adults and more often before first procedures than before subsequent procedures. More of the regimens prescribed for children exceeded the recommended duration than those prescribed for adults. Unnecessary prophylaxis was given more often when a consultant was involved than when no consultant was involved. In hospitalized patients, conformity with AHA guidelines for antimicrobial prophylaxis against endocarditis was low.

Comparison of culture, cytotoxin assay and two EIA tests with clinical diagnosis of Clostridium difficile-associated diarrhea.

OBJECTIVE: The most common etiology of infectious diarrhea in hospitalized patients is Clostridium difficile. No single laboratory test yields a definitive diagnosis. Four methods were evaluated for their sensitivity and specificity in patients who had clinically defined C difficile-associated diarrhea. METHODS: Clinical criteria for C difficile-associated diarrhea were defined. All adult in-hospital patients whose stools were tested for C difficile were prospectively followed. Stools were examined with culture on a selective medium, a commercial cytotoxicity assay (cta), and two commercially available enzyme immunoassays (eias) for toxin A (Meridian) and toxin AB (cbc). RESULTS: During the study period 235 stool specimens from 185 patients were tested. Fifty-one patients were positive for C difficile or its markers, cta was most sensitive (80%), whereas cbc-eia was most specific (98%). Differences in the sensitivities of cta and Meridian-eia were minor (80% versus 73.3%) and they were equally specific (95.5%). CONCLUSIONS: The sensitivity and specificity of eia for toxin A is similar to other tests. However, due to rapidity and ease of performance, it may be a more practical test for the diagnosis of C difficile-associated diarrhea, especially if the cytotoxin assay is not available.

The electroencephalogram in sepsis-associated encephalopathy.

To define the EEG and associated clinical features of septic encephalopathy, we studied 62 patients with positive blood cultures. Patients were divided into three clinical groups: nonencephalopathic (NE), mildly encephalopathic (ME), and severely encephalopathic (SE); the latter two groups had diffuse cerebral dysfunction. EEGs were classified into five groups: normal, excessive theta, predominant delta, triphasic waves, and suppression or burst suppression, in ascending order of severity. The EEG (1) was more sensitive than our clinical criteria for encephalopathy, (2) showed features that were, when considered with clinical and laboratory characteristics, compatible with a potentially reversible encephalopathy, and (3) had well-defined categories that correlated with percent mortality, even within a single clinical group. We conclude that the EEG is a sensitive index of brain function in septic encephalopathy and that it is especially useful in the intensive care monitoring of patients with sepsis.

Incidence of bacteremia in transesophageal echocardiography: a prospective study of 140 consecutive patients.

The incidence of bacteremia related to transesophageal echocardiography was studied in 140 consecutive patients (71 women and 69 men with a mean age of 53.7 +/- 15 years). Thirty-four patients had one or more prosthetic heart valves. Blood cultures were obtained from each patient through separate venipuncture sites immediately before and after transesophageal echocardiography. An additional late blood culture was obtained in 114 patients 1 h later. The skin was cleaned with povidone-iodine and venipunctures were performed with separate butterfly needles with use of sterile gloves and drapes. Blood samples were drawn into separate syringes, transferred to aerobic and anaerobic culture bottles and processed with use of a semiautomated system. The overall incidence of blood cultures positive for bacteremia was 2% (8 of 394 bottles) and all positive cultures grew in a single blood culture bottle. Positive cultures occurred in 4 (1.4%) of 280 bottles before the procedure, in 2 (0.7%) of 280 bottles immediately after the procedure and in 2 (0.9%) of 228 late (1-h) blood culture bottles. Bacterial isolates were coagulase-negative staphylococci (n = 5), Propionibacterium (n = 2) and Moraxella (n = 1). All were considered contaminants. Mean endoscopic time in these patients was not significantly different from that in the other patients. Follow-up of patients with a blood culture positive for bacteremia revealed no clinical evidence of systemic infection. It is concluded that 1) the incidence of bacteremia related to transesophageal echocardiography is very low, and 2) the incidence of blood cultures positive for bacteremia after transesophageal echocardiography is indistinguishable from the anticipated contamination rate.

Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis.

Dynamin was discovered in bovine brain tissue as a nucleotide-sensitive microtubule-binding protein of relative molecular mass 100,000. It was found to cross-link microtubules into highly ordered bundles, and appeared to have a role in intermicrotubule sliding in vitro. Cloning and sequencing of rat brain dynamin complementary DNA identified an N-terminal region of about 300 amino acids which contained the three consensus elements characteristic of GTP-binding proteins. Extensive homology was found between this domain and the mammalian Mx proteins which are involved in interferon-induced viral resistance, and with the product of the VPS1 locus in Saccharomyces cerevisiae, which has been implicated both in membrane protein sorting, and in meiotic spindle pole separation. Dynamin-containing microtubule bundles were not observed in an immunofluorescence study of cultured mammalian cells, but a role for a GTP-requiring protein in intermicrotubule sliding during mitosis in plants has been reported. We report here that Drosophila melanogaster contains multiple tissue-specific and developmentally-regulated forms of dynamin, which are products of the shibire locus previously implicated in endocytic protein sorting.

The encephalopathy associated with septic illness.

Physicians and surgeons have long recognized that septic illness may be accompanied by abnormal brain functions; however, no systematic, comprehensive study has been done to define the clinical and laboratory features of the syndrome of sepsis-associated encephalopathy. We undertook such a prospective study in a tertiary care hospital and found that of 69 patients with fever and microbial cultures, 32 had marked brain dysfunction, 17 showed mild encephalopathy, and 20 were clinically nonencephalopathic. Severe cases showed obtundation and paratonic rigidity while milder cases showed confusion, inappropriate behavior, inattention, disorientation, and writing errors. There were no focal neurological deficits. The following factors correlated with the severity of brain dysfunction: adult respiratory distress syndrome; fatal outcome; certain types of EEG abnormality; axonal peripheral neuropathy; elevated peripheral white blood cell count; elevated serum levels of alkaline phosphatase, bilirubin, creatinine, phosphate, potassium, and urea; reduced blood pressure and reduced serum albumin level. Our data suggest that brain functions fail with dysfunction of other organs in septic illness. Pathogenetic mechanisms are discussed. The brain dysfunction should be regarded as potentially reversible, even in severely encephalopathic cases. Prompt control of the infection is the most important measure in controlling the encephalopathy and in preventing the increased mortality found with severely encephalopathic patients.

Microbiological investigation of an outbreak of bacteraemia due to Streptococcus faecalis in an intensive care unit.

An increased incidence of bacteraemia due to Streptococcus faecalis in a Critical Care and Trauma Centre (CCTC) during November 1985 prompted investigation. During the epidemic 21 blood cultures from five CCTC patients were positive for S. faecalis. A point prevalence culture survey revealed two more strains from wounds from two of these five patients. Fifteen strains from blood cultures and the two strains from wound sites were further characterized by conventional biotyping methods and susceptibility patterns, but they could not be differentiated from 40 unrelated control strains by these methods. The API-20S and the API-ZYM systems, however, were able to distinguish the outbreak isolates from the control strains. This investigation supports the hypothesis that S. faecalis is capable of causing cross-infection, and it may be necessary to characterize enterococci beyond routine tests to identify an outbreak due to this organism.

Pulmonary cavitation due to Neisseria mucosa in a child with chronic neutropenia.

A case of spontaneous pulmonary abscess with cavitation caused by Neisseria mucosa in a chronically neutropenic child is reported. Neisseria mucosa was isolated as the sole pathogen from a percutaneous needle aspirate. It appears that the clinical course of the pulmonary lesion was indolent. Interestingly, a gallium scan was diagnostic for pulmonary abscess even though the child was neutropenic.