Molecular and antigenic characterization of a highly evolved derivative of the type 2 oral poliovaccine strain isolated from sewage in Israel

An unusual, highly diverged derivative of the Sabin type 2 oral poliovaccine (OPV) strain was recovered from environmental samples during routine screening for wild polioviruses. Virus was cultivated in L20B cells and then passaged on BGM cells at 40 degrees C (RCT [reproductive capacity at supraoptimal temperature]-positive marker) to select against most OPV strains. All but 1 of 25 RCT-positive OPV-derived environmental isolates were antigenically and genetically (>99.5% VP1 sequence match) similar to the respective Sabin strains. However, isolate PV2/4568-1/ISR98 (referred to below as 4568-1) escaped neutralization with Sabin 2-specific monoclonal antibodies and cross-adsorbed sera, and had multiple nucleotide substitutions (220 of 2,646; 8.3%) in the P1 capsid region. Fourteen of the 44 associated amino acid substitutions in the capsid mapped to neutralizing antigenic sites. Neutralizing titers in the sera of 50 Israeli children 15 years old were significantly lower to 4568-1 (geometric mean titer [GMT], 47) than to Sabin 2 (GMT, 162) or to the prototype wild strain, PV2/MEF-1/EGY42 (GMT, 108). Two key attenuating sites had also reverted in 4568-1 (A(481) to G in the 5' untranslated region and the VP1 amino acid I(143) to T), and the isolate was highly neurovirulent for transgenic mice expressing the poliovirus receptor (PVR-Tg21 mice). The extensive genetic divergence of 4568-1 from the parental Sabin 2 strain suggested that the virus had replicated in one or more people for approximately 6 years. The presence in the environment of a highly evolved, neurovirulent OPV-derived poliovirus in the absence of polio cases has important implications for strategies for the cessation of immunization with OPV following global polio eradication.     

PC-based system for collection and analysis of physiological data

A personal computer based system for data acquisition and analysis appropriate to physiological experiments is described in detail. The system is independent of the details of the analog signal generation. The software, written in C, is modular and easily portable to other PC systems. The system is capable of: (a) sampling many analog signals at an appropriate rate (100 Hz), (b) storing large quantities of digitized data, (c) analysing digitized waveforms to obtain signal parameters, and (d) storing signal parameters in a format suitable for statistical analysis. Computer processed cardiopulmonary data are compared with data derived from standard ICU equipment.     

Graft-versus-host disease as adoptive immunotherapy in patients with advanced hematologic neoplasms

The occurrence of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation for leukemia is thought to decrease the probability of recurrence. To study this effect (called adoptive immunotherapy) we modified the prophylaxis of GVHD in patients with advanced hematologic neoplasms (mostly leukemia) who received bone marrow transplants. Patients under 30 years of age were randomly assigned to one of three regimens of post-transplantation immunosuppression: Group I (n = 44) received a standard course of methotrexate for 102 days after transplantation, Group II (n = 40) received an abbreviated (11-day) course of methotrexate, and Group III (n = 25) received the standard course of methotrexate plus viable buffy-coat cells from the marrow donors. All 109 patients received cyclophosphamide (60 mg per kilogram of body weight on each of two days), total-body irradiation (2.25 Gy daily for seven days), and unmodified marrow from HLA-identical sibling donors. The frequency of GVHD of Grades II through IV was 25 percent in Group I, 59 percent in Group II, and 82 percent in Group III (P = 0.0001). The incidence of chronic GVHD, however, did not differ significantly among the groups (33, 51, and 44 percent, respectively), nor did the five-year probability of recurrence of disease (38, 45, and 33 percent, respectively). However, mortality from causes other than cancer was 34 percent in Group I, 45 percent in Group II, and 64 percent in Group III (I vs. III, P = 0.024); the deaths were due primarily to infections complicating the course of GVHD. With a median follow-up of 5.1 years (range, 3.9 to 7.4), disease-free survival was 41 percent in Group I, 30 percent in Group II, and 24 percent in Group III (the differences were not statistically significant). We conclude that abbreviating methotrexate prophylaxis or infusing donor buffy-coat cells increased the incidence of acute GVHD and related mortality without altering the incidence of chronic GVHD or the recurrence of malignant disease.     

Medication adherence in patients with HIV infection: a comparison of two measurement methods

Two measurements of adherence, patient self-report and electronic measurement by the Medication Event Monitoring System (MEMS), were compared in a 3-month adherence study of 44 HIV-infected patients who had been placed on regimens that included protease inhibitors (PIs). The dose percentage and degree of clinically significant dosing time fluctuation were calculated monthly. The mean dose percentage by self-report versus MEMS was 97.5% versus 90.3% during month 1 of adherence monitoring, 96.5 versus 90.1% during month 2, and 98.4% versus 92.8% during month 3. Thirty-two percent of patients taking PIs and 21% of patients taking nucleoside analogues demonstrated clinically significant dosing time fluctuation. Our data confirm that self-reports of adherence overestimate true adherence behavior, and patients' self-reports of dosing times may not accurately reflect their deviation from those times.     

A simple breathing circuit to maintain isocapnia during measurements of the hypoxic ventilatory response

We report the development and testing of a simple breathing circuit that maintains isocapnia in human subjects during hypoxic hyperpnea. In addition, the circuit permits rapid switching between two gas mixtures with different partial pressures of oxygen. Eleven volunteers breathed repeated cycles of exposure to air (2 min of 21% O(2), balance N(2)) and hypoxia (2 min of 8.3+/-0.1% O(2), balance N(2)). Hypoxia induced significant increases in minute ventilation, breathing frequency and tidal volume (P < 0.05) that were consistent over repeated cycles of hypoxia (P > 0.1, one-way ANOVA). The system successfully maintained isocapnia in all subjects, with an average change in end-tidal CO(2) of only -0.2 mmHg during hyperventilation in hypoxia (range 0.4 to -0.8 mmHg). This system may be suitable for repeated tests of the hypoxic ventilatory response (HVR) and may prove useful for exploring intra- and inter-individual variability of HVR in humans.     

Transmission of human cytomegalovirus from infected uterine microvascular endothelial cells to differentiating/invasive placental cytotrophoblasts

Analysis of placentas infected with human cytomegalovirus (CMV) suggested that viral transmission could involve differentiating/invasive cytotrophoblasts in villi that attach the placenta to the uterine wall. To parse the cellular components in this process, we developed a coculture system of polarized uterine microvascular endothelial cell (UtMVEC) infection with an endothelial cell-tropic pathogenic strain of CMV. Then we evaluated the potential role of neutrophils and endothelial cells in the spread of infection to differentiating cytotrophoblasts. As shown by immunocytochemistry and analysis of viral replication, CMV preferentially infected endothelial cells via apical membranes and disrupted cell junction proteins, thereby altering paracellular permeability and cell polarity. Neutralizing antibodies to CMV glycoprotein B, an envelope component that facilitates virion penetration, blocked plaque formation in polarized UtMVEC. Neutrophils transmitted CMV infection to UtMVEC, which in turn infected cytotrophoblasts. However, neutrophils did not directly infect cytotrophoblasts. These findings implicate endothelial cells from the uterine microvasculature as a potential source for CMV infection of endovascular cytotrophoblasts of the anchoring villi. Possibly the cytokine/chemokine milieu in the pregnant uterus could attract immune cells that infect endothelial cells in hybrid fetal-maternal vessels. In turn, these cells could infect endovascular cytotrophoblasts, one possible initiation point of a cascade that results in retrograde placental CMV infection.     

Immunoprophylaxis of Chlamydia trachomatis lymphogranuloma venereum pneumonitis in mice by oral immunization.

Groups of BALB/c mice were orally immunized with chlamydiae and challenged intranasally to determine whether oral immunization offers protection against pulmonary disease and to characterize the nature and kinetics of the chlamydial antibody response in the lung and other mucosal sites. Protection by oral immunization from chlamydial lung disease was demonstrated by lack of replication of the organism and the lack of chlamydial antigen in lung tissue. The chlamydial immunoglobulin A (IgA) antibody response was present at all body sites, reaching peak levels in the lung as well as in the serum. Classical IgA booster effect kinetics was observed after intranasal challenge, especially in the lung. Specific IgG antibody was detected at all body sites but at lower levels. Furthermore, animals immunized orally had no pneumonic process, as determined by histopathology. These studies also suggest that passively acquired specific serum IgG antibody may not significantly influence the course of mucosal replication of the organism. These observations indicate that oral immunization activating the gut-associated lymphoid tissue system gave total protection against chlamydial lung disease, suggesting migration of immunologically competent cells from the intestine to the lung.     

Defects in neurofibromatosis 2 protein function can arise at multiple levels

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome resulting from mutations in the NF2 tumor suppressor gene. Analysis of NF2 mutations has revealed some general genotype-phenotype correlations. Severe disease has been associated with mutations that produce a premature termination while more mild disease has been associated with missense mutations. Here, we provide experimental proof for these genotype-phenotype correlations by demonstrating that nonsense mutations fail to produce stable merlin protein while missense mutations result in the generation of merlin proteins defective in negative growth regulation. This inability to suppress cell growth may result from defects in the function of merlin at several levels, including failure to form an intramolecular complex. Based on these findings, we propose a model for merlin growth suppression that provides a framework for analyzing NF2 patient mutations and merlin function.      

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.