Suppression of HIV-1 infection in primary CD4 T cells transduced with a self-inactivating lentiviral vector encoding a membrane expressed gp41-derived fusion inhibitor.

Peptidomimetics of HIV-1 gp41 sequences required for membrane fusion are potent inhibitors of HIV-1 entry. We hypothesize that expression of a membrane-bound gp41-derived fusion inhibitor will confer HIV-1 resistance to primary CD4 T cells. Efficient gene delivery and stable expression of a membrane-bound gp41-derived fusion inhibitor to primary CD4 T cells was accomplished using a self-inactivating lentiviral vector. A potent antiviral effect was observed when transduced CD4 T cells were challenged with a highly virulent CXCR4-tropic strain of HIV-1. Production of soluble p24 in the supernatant was inhibited 100-fold, and cytopathic effects were evident early in non-transduced cells and absent in transduced cells. Expression of the gp41 sequences was not detrimental to CD4 cells as transduced CD4 T cells exhibited a population doubling time that was equivalent to T cells transduced with a control vector. Results from this study support the rationale to use this lentiviral vector targeted at HIV entry as a potential gene therapy for HIV infection.

A controlled trial of topical nitroglycerin in a New Zealand white rabbit model of brown recluse spider envenomation.

STUDY OBJECTIVES: Topical nitroglycerin has been reported to prevent skin necrosis from brown recluse spider bites, but this has never been scientifically tested. This study attempts to assess the effects of topical nitroglycerin on experimental Loxosceles reclusa envenomations. METHODS: We performed a randomized, blinded, controlled study in an animal care facility. Twenty-four New Zealand white rabbits were experimentally envenomated by means of subcutaneous injection with 20 microg of brown recluse spider venom. Rabbits were randomized to 1 of 2 experimental groups. The treatment group received 1 in of 2% topical nitroglycerin ointment every 6 hours for 3 days applied directly to the envenomation site. The control group received the vehicle without nitroglycerin. Gross examination of the lesions and measurements of the areas of the lesions were made daily. Creatine phosphokinase (CPK), blood urea nitrogen, creatinine, hemoglobin, and hematocrit levels were measured on days 0, 5, and 10. Lesions were excised after 10 days and examined by a blinded pathologist, who measured the area of necrosis and quantified inflammation and edema using a standard wound-healing score. For all values, mean values plus SD were determined. All comparisons made over multiple time points were assessed for significance by using a repeated-measures analysis of variance followed by Fisher least significant difference and Scheffé post hoc comparisons. A P value of.05 or less was used to determine significance. The Student's t test was used to compare the means of single measures. Significance was determined by using 95% confidence intervals. Comparisons of total area of necrosis were made with the nonparametric Mann-Whitney U test because of the heavy positive skew of the data. RESULTS: Skin necrosis developed in all animals. Mean values of the lesion area were not significantly different over time between the 2 groups of animals. At day 10, the median area of necrosis was 22.3 cm2 for the treatment group and 15.4 cm2 for the control group (P =.12). The inflammation score was 3.33+/-0.78 for the treatment group and 2.79+/-1.29 for the control group (P < .01). The edema score was 1.25+/-1.28 for the treatment group and 0.98+/-1.10 for the control group (not significantly different). CPK levels increased dramatically in both groups, with the greatest increase in the treatment group. In both groups hemoglobin and hematocrit levels decreased significantly, whereas WBC counts and platelet counts increased significantly, without significant differences between the 2 groups. CONCLUSION: At the dose used in this experiment, topical nitroglycerin did not prevent skin necrosis, increased inflammation score, and increased serum CPK levels. The results of this study do not support the use of topical nitroglycerin in the treatment of L reclusa envenomation and suggest that systemic toxicity could be increased.

Cardiovascular pressure-flow relationships: what should be taught?

Cardiovascular physiology reflects the interrelations of flow, pressure, and resistance. Undergraduate students are often confused by the complexity of the system. This symposium presents a sequential presentation of the underlying concepts, building on analogies, past experience, and conceptual models to allow students to develop a physiologically appropriate understanding of cardiovascular physiology.

Design and evaluation of a national set of learning objectives: the medical physiology learning objectives project.

In 1998, the American Physiological Society (APS) and the Association of Chairs of Departments of Physiology (ACDP) began collaboration on a project to develop a set of physiology learning objectives for medical students. Over the next 2 years, more than 50 physiologists collaborated in the development a comprehensive draft containing its 695 learning objectives. Faculty in 31 medical schools in the United States, Canada, and Puerto Rico evaluated these objectives. On the basis of this evaluation, the ACDP recommended deleting 13 of them. The final project, containing 682 objectives, was approved in December 2000 by the ACDP and published on the APS website http://www.the-aps.org/education/MedPhysObj/medcor.htm. The identification of the "content" of medical physiology instruction provides the APS and the ACDP with a tool to introduce emerging topics, such as the physiology of aging and gender differences, at a national level. The medical physiology learning objectives project provides a guide for directing current and future medical physiology instruction in the United States.

Modulation of susceptibility to HIV-1 infection by the cytotoxic T lymphocyte antigen 4 costimulatory molecule.

CD4 T cells activated in vitro by anti-CD3/28-coated beads are resistant to infection by CC chemokine receptor 5 (CCR5)-dependent HIV-1 isolates. In vivo, antigen-presenting cells (APCs) activate CD4 T cells in part by signaling through the T cell receptor and CD28, yet cells stimulated in this manner are susceptible to HIV-1 infection. We show that cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement counteracts the CD28 antiviral effects, and that the ratio of CTLA-4 to CD28 engagement determines the susceptibility of HIV-1 infection. Furthermore, unopposed CTLA-4 signaling provided by CD28 blockade promotes vigorous HIV-1 replication, despite minimal T cell proliferation. Finally, CTLA-4 antibodies decrease the susceptibility of antigen-activated CD4 T cells to HIV, suggesting a potential approach to prevent or limit viral spread in HIV-1-infected individuals.

Reduced oxygen consumption precedes the drop in body core temperature caused by hemorrhage in rats.

This study examines the early time course in core temperature change and oxygen consumption at 4 levels of hemorrhage. Chronically instrumented rats were acclimatized to a respirometry chamber for 30 min. The rats were briefly (10 min) removed from the chamber for a fixed volume hemorrhage of 0 mL/kg (sham), 8 mL/kg, 16 mL/kg, 24 mL/kg, or 32 mL/kg. Rats were then returned to the chamber, and oxygen consumption and body core temperature were monitored for the next 2 h. Oxygen consumption (control 1.26 mL O2/g/h) fell significantly 5 min after hemorrhage in all but the sham and 8 mL/kg hemorrhage groups, with the decrease proportional to the hemorrhage volume. The 32 mL/kg hemorrhage group showed the greatest decrease, to 0.47 mL O2/g/h. Body core temperature (control 37.5 degrees C) fell more gradually, declining to 35.6 degrees C 110 min after the 24 mL/kg hemorrhage, and to 33.2 degrees C at 6 h after the 32 mL/kg hemorrhage. In the 16 mL/kg hemorrhage group, oxygen consumption fell significantly by 5 min after hemorrhage, but a drop in body temperature was not seen until 25 min after hemorrhage. The data from this study indicate that the drop in core temperature does not cause the observed decrease in oxygen consumption. In fact, the timing and magnitude of the drop in oxygen consumption indicate that the reduced metabolic rate may mediate the hemorrhage-induced drop in body core temperature in conscious rats.

Reduced metabolic rate accompanies the hemorrhage-induced hypothermia in conscious rats.

The mechanism for the hemorrhage-induced drop in body temperature is unknown. This study determined the alterations in cutaneous heat exchange and metabolic heat production caused by a moderate hemorrhage in conscious rats. Chronically instrumented rats were subjected to a 16 ml/kg hemorrhage, followed by a 4-h recovery period, while monitoring body core temperature and cutaneous temperature. Cutaneous heat transfer was disrupted by housing the animals at an elevated (28 degrees C) ambient temperature. A separate group of experiments measured the change in oxygen consumption in the post-hemorrhage period. Moderate hemorrhage caused a drop in body core temperature which stabilized at 0.7+/-0.3 degrees C below control in the second hour following hemorrhage. Disruption of cutaneous heat exchange by reducing the thermal gradient did not diminish the hemorrhage-induced hypothermia. Hemorrhage caused a significant decline of oxygen consumption (-0. 21+/-0.05 ml O(2)/g per h). This 16% drop in resting oxygen consumption was prevented by immediately retransfusing the aspirated blood back into the rat. These data indicate that a decrease in metabolic heat production mediates the drop in body core temperature caused by moderate hemorrhage in conscious rats.

Influenza virus upregulates CXCR4 expression in CD4+ cells.

We examined the effect of prior influenza virus infection on the susceptibility of CD4+ cells to HIV-1 infection. Influenza virus infection of HeLa-CD4 cells resulted in a marked increase in susceptibility to infection by CXCR4-dependent but not CCR5-dependent HIV isolates. Influenza virus infection resulted in an increase in the steady state level of CXCR4 transcripts and an increase in cell surface CXCR4 expression. Our observations suggest that infectious agents such as influenza may contribute to HIV disease progression by modulating coreceptor availability.

Quantitation of HIV-1 Entry Cofactor Expression.

Infection of CD4+ T-lymphocytes by HIV-1 is initiated by binding of the virus to the CD4 receptor on the lymphocyte surface, followed by fusion of the virus with the cell membrane (1). However, expression of CD4 on certain non-human cells does not render them permissive for HIV-1 entry, suggesting that an additional entry cofactor is required (2,3). The identity of this cofactor remained elusive until Berger and colleagues reported that the α-chemokine receptor fusin/CXCR4 functions as the entry cofactor for T-cell line-tropic isolates of HIV-1 (4). Subsequent progress in the field has been rapid. To date, three ß-chemokine receptors (CCR2b, CCR3, and CCR5) have also been identified as HIV-1 entry cofactors (5).

A comparison of demand-valve and bag-valve ventilations in a swine pneumothorax model.

OBJECTIVE: Two means of delivering artificial ventilation readily available to out-of-hospital personnel are the bag-valve (BV) and the O2-powered demand-valve (OPDV). However, use of the OPDV has been limited because of concerns that it may worsen an underlying pneumothorax. This study compared the changes in size of pneumothorax in swine ventilated with the 2 devices. METHODS: Three swine were anesthetized, intubated, and instrumented with a femoral arterial line and a pediatric Swan-Ganz catheter. A chest tube was placed, the chest was opened, and the lung parenchyma was visualized. The lung was disrupted by a single stab with a #10 scalpel; the chest was then sealed; and a pneumothorax was created by injecting 30 mL of air through the chest tube. The animals were ventilated by 12 emergency medical technicians using either BV or OPDV. After 10 minutes of ventilation, the pneumothorax volume was measured. RESULTS: When comparing final pneumothorax volumes after 10 minutes of ventilation with the 2 devices, there was no significant difference (mean +/- SD = 40.8 +/- 28.2 mL vs 52.3 +/- 23.1 mL, p = 0.286). CONCLUSION: There is no difference in final pneumothorax volumes after OPDV or BV ventilation.

Large-scale production of CD4+ T cells from HIV-1-infected donors after CD3/CD28 costimulation.

We describe a procedure for large-scale enrichment, growth, and harvesting CD4+ T cells. This method may be effective for HIV-1 immunotherapy, as the mode of stimulation, with anti-CD3 plus anti-CD28 coated beads (CD3/CD28 beads) induces a potent antiviral effect. PBMC were obtained by density gradient centrifugation of an apheresis product. Monocytes/macrophages were removed by incubating PBMC with beads coated with IgG. The cells were then magnetically depleted of B cells and CD8+ cells with mouse anti-CD20 and anti-CD8 MAbs and sheep antimouse coated beads. The remaining cells were >80% CD4+ and were transferred to gas-permeable bags containing CD3/CD28 beads and cultured in a closed system. After 14 days, the cell number increased an average of 37-fold, and cells were nearly 100% CD4+. Viral load, assessed by DNA PCR for HIV-1 gag, decreased >10-fold during culture in the absence of antiretroviral agents. Removal of CD3/CD28 beads from the cell suspension was accomplished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep magnetic separator using gravity-driven flow. The cells were then concentrated to 300 ml in an automated centrifuge. This process allows safe and efficient growth of large numbers of CD4+ T cells from HIV-1+ donors.

Tat-associated kinase, TAK, activity is regulated by distinct mechanisms in peripheral blood lymphocytes and promonocytic cell lines.

TAK, a multisubunit cellular protein kinase that specifically associates with the human immunodeficiency virus Tat proteins and hyperphosphorylates the carboxyl-terminal domain of RNA polymerase II, is a cofactor for Tat and mediates its transactivation function. The catalytic subunit of TAK has been identified as cyclin-dependent kinase Cdk9, and its regulatory partner has been identified as cyclin T1; these proteins are also components of positive transcription elongation factor P-TEFb. TAK activity is up-regulated upon activation of peripheral blood lymphocytes and following macrophage differentiation of promonocytic cell lines. We have found that activation of peripheral blood lymphocytes results in increased mRNA and protein levels of both Cdk9 and cyclin T1. Cdk9 and cyclin T1 induction occurred in purified CD4(+) primary T cells activated by a variety of stimuli. In contrast, phorbol ester-induced differentiation of promonocytic cell lines into macrophage-like cells produced a large induction of cyclin T1 protein expression from nearly undetectable levels, while Cdk9 protein levels remained at a constant high level. Measurements of cyclin T1 mRNA levels in a promonocytic cell line suggested that regulation of cyclin T1 occurs at a posttranscriptional level. These results suggest that cyclin T1 and TAK function may be required in differentiated monocytes and further show that TAK activity can be regulated by distinct mechanisms in different cell types.

Naïve and memory CD4 T cells differ in their susceptibilities to human immunodeficiency virus type 1 infection following CD28 costimulation: implicatip6s for transmission and pathogenesis.

In vitro evidence suggests that memory CD4(+) cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA+ CD4(+) (naïve) and CD45RO+ CD4(+) (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4(+) cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4(+) subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta upon stimulation. Neutralization of these beta-chemokines rendered memory CD4(+) cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and beta-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4(+) T cells serve as the targets for early rounds of HIV-1 replication.

Sentinel lymph node localization in early breast cancer.

METHODS: Thirty-two patients with clinical node-negative breast cancer underwent sentinel node localization study as part of a National Cancer Institute-sponsored multicenter trial. Anatomical and histopathologic characteristics of sentinel lymph node (SLN) and a kinetic analysis of nodal uptake were studied. Patients were injected with 1 mCi/4 ml unfiltered 99mTc-sulfur colloid in four divided doses around the palpable lesion or immediately adjacent to the excision cavity if prior biopsy was performed. SLN biopsy was performed 1.5-6 hr (mean = 3 hr) postinjection. Intraoperative localization was performed using a gamma probe. All patients underwent complete axillary dissection. RESULTS: SLN was identified in 30 of 32 (94%) patients. There were no false-negative SLN biopsies. CONCLUSION: This study supports the clinical validity of SLN biopsy in breast cancer and confirms that, unlike the blue dye technique, the learning curve with unfiltered 99mTc-sulfur colloid and the gamma detection probe is short, and SLN localization is achievable in over 90% of cases by surgeons with modest experience. The use of unfiltered 99mTc-sulfur colloid (larger particle size) with larger injected volume permits effective localization of SLNs.

The role of co-stimulation in regulation of chemokine receptor expression and HIV-1 infection in primary T lymphocytes.

Fusion and entry of the human immunodeficiency virus (HIV) into CD4(+) T lymphocytes requires expression of CD4 and a coreceptor. At least eight chemokine receptors can serve as coreceptors for HIV. Accumulating evidence indicates that multiple factors, including the state of cellular differentia- tion and activation, regulate the expression of alpha- and beta-chemokine receptors on lymphocytes. For example, binding of antibodies to the CD28 coreceptor can downregulate expression of beta-chemokine receptors, and this appears to have important consequences on the susceptibility of CD4(+) T lymphocytes to infection by HIV-1. In contrast, binding of the natural CD28 ligand B7 or antibodies to the CD28 homologue CTLA-4 can upregulate CCR5 expression, sug- gesting a reciprocal interaction between CD28 and CTLA-4 and the regulation of beta-chemokine receptor expression. Thus, the CD28/CTLA-4/B7 co-stimulation pathway is identi- fied as a potential novel target for the control of susceptibility to some strains of HIV-1 infection.

Productive infection of neonatal CD8+ T lymphocytes by HIV-1.

CD8+ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8+ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell-tropic (T-tropic) HIV strains. Physiological activation of CD8-alpha/beta+ CD4- T cell receptor-alpha/beta+ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1-activated cord blood CD8+ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8+ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.

Ventilatory strategies affect gas exchange in a pig model of closed-chest cardiac compression.

STUDY OBJECTIVE: To identify the arterial and mixed venous blood gas changes caused by different ventilatory strategies during resuscitation from ventricular fibrillation in a pig model of closed-chest cardiac compression. METHODS: A prospective randomized animal study was performed using 27 domestic pigs (body weight, 30 to 35 kg). Pentobarbital-anesthetized pigs were assigned to receive one of three treatments: (1) chest compression without assisted ventilation (n = 8), (2) assisted ventilation with room air (n = 8), and (3) assisted ventilation with 100% oxygen (n = 8). A fourth group, with the airway completely blocked, was added at the end of the experiment (n = 3). After instrumentation, the ventricles were fibrillated, and chest compression was begun 30 seconds after fibrillation with the use of the Thumper Mechanical CPR system (Michigan Instruments). Arterial and mixed venous blood gas samples were collected at 1, 3, 10, and 20 minutes of resuscitation. Defibrillation was attempted after the 20-minute sample was taken. RESULTS: Fibrillation followed by chest compression alone caused a significant drop in arterial and mixed venous partial pressure of oxygen (PO2) and a significant increase in arterial and mixed venous partial pressure of carbon dioxide (PCO2). Compared with the chest compression only, ventilation with room air significantly increased arterial and mixed venous PO2 and decreased arterial and mixed venous PCO2. Ventilation with 100% oxygen further increased arterial and mixed venous PO2 but did not affect PCO2, when compared with room air ventilation. The only successful defibrillations (3 animals) occurred in the group receiving 100% oxygen. CONCLUSION: This study indicates that passive air movement during chest compression does not allow physiologically significant pulmonary gas exchange and that room air ventilation alone is not sufficient to maintain mixed venous PO2.

Current and future impact of technology on physiology education.

There are at least three areas in which technology can impact education: teaching, learning, and assessment. Teaching, when viewed as communication of information, has been transformed by the technology revolution. Word processing, multimedia, distance learning, and access to the World Wide Web are some prominent examples. The impact of technology on learning, defined as knowledge or skill acquired by instruction or study, has been less dramatic, in part because of our limited understanding of cognitive processes. Some forms of assessment, the collection of evidence of learning, have benefited from technology, such as item analysis of multiple-choice questions. To be effective, the focus on instruction must start with the learner and, from there, consider what should be done to enhance learning. An emphasis on what is technologically appropriate, rather than what is technologically possible, will improve the quality of both teaching and learning.

Gamma probe guided sentinel node biopsy in breast cancer.

Axillary lymph node status is the most important pathological determinant of prognosis in early breast cancer. Determination of axillary status is crucial in clinical decision-making. It is currently accepted that the total axillary lymphadenectomy is the most reliable staging procedure. However, routine axillary dissection does not benefit a majority of early breast cancer patients who are node-negative, and the patients sustain the potential morbidity and the economic cost of this procedure. There is substantial evidence that there is an orderly progression of breast cancer metastases in a lymphatic basin, sentinel node being the first node to receive lymphatic drainage from the tumor site. Sentinel lymph node biopsy may prove to be the optimal sampling technique for staging of breast cancer patients. A large multicenter trial to study the clinical validity of sentinel lymph node biopsy in breast cancer is underway. This paper addresses the rationale for sentinel lymph node biopsy and discusses the technical issues with regard to anatomy and physiology of breast lymphatics.