Preclinical targeting of aggressive T-cell malignancies using anti-CD5 chimeric antigen receptor.

The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.

Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells.

Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.

Evaluation of the dynamic cutoff rigidity model using dosimetry data from the STS-28 flight.

We have a developed a dynamic cutoff rigidity model based on computed world grids of vertical cutoff rigidities derived from employing the Tsyganenko magnetospheric model. The dynamic range of this model covers all magnetic activity levels specified by integer values of the Kp magnetic index. We present comparisons of the measured dose observed on the space shuttle during the August 1989 solar proton event with the dose computed from solar particles predicted to be allowed through the magnetosphere to the space shuttle position. We find a one-to-one correspondence between the portion of the orbit predicted to be subjected to solar protons and the portion of the orbit where solar particle dose measurements were obtained.

Vitronectin is sequestered within human spermatozoa and liberated following the acrosome reaction.

Vitronectin plays a role in the regulation of complement and thrombin activities and in cell surface proteolysis. Vitronectin is also an intrinsic protein of human spermatozoa. Vitronectin message has been detected in whole testis poly-A mRNA and localized by in-situ reverse transcription-polymerase chain reaction to spermatocytes. The proportion of spermatozoa that express vitronectin increases following their capacitation. In this study, spermatozoa from a man of proven fertility were probed with an anti-vitronectin monoclonal antibody (VN7) before and after their permeabilization with 0.1% Triton X-100. Of fresh spermatozoa observed by confocal microscopy, 0-8% showed vitronectin staining. However, 75% of those observed displayed vitronectin following permeabilization. Serial confocal sections through the sperm head confirmed the internal localization of vitronectin. The acrosomal status of capacitated spermatozoa that expressed vitronectin was then determined. Dual colour microscopy with rhodamine-conjugated anti-vitronectin antibody and a fluorescein-conjugated antibody directed against CD46 (a complement regulatory protein expressed on the inner acrosomal membrane) revealed that only acrosome-reacted (CD46-positive) spermatozoa displayed vitronectin. Two populations of these spermatozoa were observed. Fifty-seven of 260 (22%) were CD46-positive/vitronectin-positive and 72 of 260 (28%) were CD46-positive/vitronectin-negative. No spermatozoa were CD46-negative/vitronectin-positive. These results confirm that vitronectin is released from a sequestered location within the spermatozoon following the acrosome reaction.

Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining.

The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa.

Disparity between serological reactivity to Borrelia burgdorferi and evidence of past disease in a high-risk group.

A prevalence study of past Lyme borreliosis in persons with outdoor occupations was done. Consenting individuals (n = 302) were administered a questionnaire eliciting demographic and occupational data and a clinical history, and were asked to donate a serum specimen for detection of antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and borrelia inhibition assays, and for detection of potentially cross-reactive antibodies. Of 302 individuals, 77 (25%) had reactive antibodies detected by ELISA. Of these 302 individuals, 44 (15%) met the criteria of the Centers for Disease Control and Prevention for serological reactivity as evidenced by immunoblotting, and 70 (23%) had inhibitory activity. Through the clinical criteria employed, only 11 individuals with serological reactivity had prior illness compatible with Lyme borreliosis. Higher ELISA absorbances were positively correlated with age and duration of outdoor occupation. The results from three serological assays and the lack of reactivity to potentially cross-reactive infectious agents indicate that serological reactivity was due to exposure to B. burgdorferi. The disparity between serological reactivity and the clinical evidence of Lyme borreliosis suggests cumulative exposure to a nonpathogenic form of B. burgdorferi.

Lyme borreliosis: laboratory considerations.

The laboratory is frequently the primary source for making the diagnosis of Lyme borreliosis when the symptoms are vague and the clinical hallmarks are missing. However, the temptation to interpret the laboratory results out of context from the clinical history and presentation should be avoided since the laboratory tests for evidence of B. burgdorferi infection can also be problematic. These problems have included sensitivity/specificity problems, lack of standardization of both methology and interpretation, and lack of routine direct evidence (ie culture) testing. These problems are discussed as well as possible solutions. It should be noted that meaningful results can usually be obtained from these tests if caution is used in their interpretation and the clinical symptomatology, history, and examination are integrated with the laboratory results.

Measurements of the linear energy transfer spectra on the Mir orbital station and comparison with radiation transport models.

A tissue equivalent proportional counter designed to measure the linear energy transfer spectra (LET) in the range 0.2-1250 keV/micrometer was flown in the Kvant module on the Mir orbital station during September 1994. The spacecraft was in a 51.65 degrees inclination, elliptical (390 x 402 km) orbit. This is nearly the lower limit of its flight altitude. The total absorbed dose rate measured was 411.3 +/- 4.41 microGy/day with an average quality factor of 2.44. The galactic cosmic radiation (GCR) dose rate was 133.6 microGy/day with a quality factor of 3.35. The trapped radiation belt dose rate was 277.7 microGy/day with an average quality factor of 1.94. The peak rate through the South Atlantic Anomaly was approximately 12 microGy/min and nearly constant from one pass to another. A detailed comparison of the measured LET spectra has been made with radiation transport models. The GCR results are in good agreement with model calculations; however, this is not the case for radiation belt particles and again points to the need for improving the AP8 omni-directional trapped proton models.

In-flight radiation measurements on STS-60.

A joint investigation between the United States and Russia to study the radiation environment inside the Space Shuttle flight STS-60 was carried out as part of the Shuttle-Mir Science Program (Phase 1). This is the first direct comparison of a number of different dosimetric measurement techniques between the two countries. STS-60 was launched on 3 February 1994 in a nearly circular 57 degrees x 353 km orbit with five U.S. astronauts and one Russian cosmonaut for 8.3 days. A variety of instruments provided crew radiation exposure, absorbed doses at fixed locations, neutron fluence and dose equivalent, linear energy transfer (LET) spectra of trapped and galactic cosmic radiation, and energy spectra and angular distribution of trapped protons. In general, there is good agreement between the U.S. and Russian measurements. The AP8 Min trapped proton model predicts an average of 1.8 times the measured absorbed dose. The average quality factor determined from measured lineal energy, y, spectra using a tissue equivalent proportional counter (TEPC), is in good agreement with that derived from the high temperature peak in the 6LiF thermoluminescent detectors (TLDs). The radiation exposure in the mid-deck locker from neutrons below 1 MeV was 2.53 +/- 1.33 microSv/day. The absorbed dose rates measured using a tissue equivalent proportional counter, were 171.1 +/- 0.4 and 127.4 +/- 0.4 microGy/day for trapped particles and galactic cosmic rays, respectively. The combined dose rate of 298.5 +/- 0.82 microGy/day is about a factor of 1.4 higher than that measured using TLDs. The westward longitude drift of the South Atlantic Anomaly (SAA) is estimated to be 0.22 +/- 0.02 degrees/y. We evaluated the effects of spacecraft attitudes on TEPC dose rates due to the highly anisotropic low-earth orbit proton environment. Changes in spacecraft attitude resulted in dose-rate variations by factors of up to 2 at the location of the TEPC.

Results of time-resolved radiation exposure measurements made during U.S. Shuttle missions with a tissue equivalent proportional counter.

Time-resolved radiation exposure measurements inside the crew compartment have been made during recent Shuttle missions with the USAF Radiation Monitoring Equipment-III (RME-III), a portable four-channel tissue equivalent proportional counter. Results from the first six missions are presented and discussed. The missions had orbital inclinations ranging from 28.5 degrees to 57 degrees, and altitudes from 200-600 km. Dose equivalent rates ranged from 40-5300 micro Sv/dy. The RME-III measurements are in good agreement with other dosimetry measurements made aboard the vehicle. Measurements indicate that medium- and high-LET particles contribute less than 2% of the particle fluence for all missions, but up to 50% of the dose equivalent, depending on the spacecraft's altitude and orbital inclination. Iso-dose rate contours have been developed from measurements made during the STS-28 mission. The drift rate of the South Atlantic Anomaly (SAA) is estimated to be 0.49 degrees W/yr and 0.12 degrees N/yr. The calculated trapped proton and Galactic Cosmic Radiation (GCR) dose for the STS-28 mission were significantly lower than the measured values.

Cell-mediated immunity in allopurinol-induced hypersensitivity.

Allopurinol may induce severe hypersensitivity characterized by hepatitis, interstitial nephritis, and skin rash. The mechanisms for this hypersensitivity syndrome are incompletely elucidated. Immunologic studies were performed on tissue and peripheral blood lymphocytes from a patient with allopurinol hypersensitivity. Immunohistochemistry was performed on sections of the liver biopsy utilizing monoclonal antibodies for T and B lymphocytes. Peripheral blood lymphocyte immunophenotyping by flow cytometry and peripheral blood lymphocyte stimulation studies with either allopurinol or oxypurinol measured as tritiated thymidine uptake were performed in the hypersensitive patient and compared to a group of six patients treated with allopurinol without hypersensitivity and eight normal control patients. Additional single- and dual-color immunophenotyping by flow cytometry of oxypurinol-stimulated lymphocytes was performed in the hypersensitive patient and compared to normal controls. The liver biopsy demonstrated predominantly a T lymphocyte infiltrate. The number of peripheral blood lymphocytes expressing activation antigens was significantly greater in the hypersensitive patient compared to that of both control groups. Lymphocytes from the hypersensitive patient were moderately stimulated by allopurinol and markedly stimulated by oxypurinol compared to both control groups. Oxypurinol-stimulated lymphocytes from the hypersensitive patient demonstrated enhanced expression of activation antigens compared to unstimulated lymphocytes from this patient and normal controls. These studies suggest that cell-mediated immunity directed toward allopurinol and more importantly to its oxypurinol metabolite is involved in the pathogenesis of allopurinol-induced hypersensitivity.

Radiation dosimetry measurements during U.S. Space Shuttle missions with the RME-III.

Time-resolved radiation dosimetry measurements inside the crew compartment have been made during recent Shuttle missions with the U.S. Air Force Radiation Monitoring Equipment-III (RME-III), a portable battery-powered four-channel tissue equivalent proportional counter. Results from the first six missions are presented and discussed. Half of the missions had orbital inclinations of 28.5 degrees with the remainder at inclinations of 57 degrees or greater; altitudes ranged from 300 to 600 km. The determined dose equivalent rates ranged from 70 to 5300 microSv/day. The RME-III measurements are in good agreement with other dosimetry measurements made aboard the vehicles. Measurements indicate that medium- and high-LET particles contribute less than 2% of the particle fluence for all missions, but up to 50% of the dose equivalent, depending on the spacecraft's altitude and orbital inclination. Isocontours of fluence, dose and dose equivalent rate have been developed from measurements made during the STS-28 mission. The drift rate of the South Atlantic Anomaly is estimated to be 0.49 degrees W/yr and 0.12 degrees N/yr. The calculated trapped proton and GCR dose for the STS-28 mission was significantly lower than the measured values.

Analysis of radiation risk from alpha particle component of solar particle events.

The solar particle events (SPE) will contain a primary alpha particle component, representing a possible increase in the potential risk to astronauts during an SPE over the often studied proton component. We discuss the physical interactions of alpha particles important in describing the transport of these particles through spacecraft and body shielding. Models of light ion reactions are presented and their effects on energy and linear energy transfer (LET) spectra in shielding discussed. We present predictions of particle spectra, dose, and dose equivalent in organs of interest for SPE spectra typical of those occurring in recent solar cycles. The large events of solar cycle 19 are found to have substantial increase in biological risk from alpha particles, including a large increase in secondary neutron production from alpha particle breakup.

Occurrence of antibodies to Borrelia burgdorferi in patients with nonspirochetal subacute bacterial endocarditis.

OBJECTIVE: To determine the prevalence and specificity of antibodies to Borrelia burgdorferi in patients with nonspirochetal subacute bacterial endocarditis and assess whether increased levels of antibodies to B. burgdorferi were attributable to rheumatoid factor. DESIGN: Retrospective case-control study. SETTING: Urban referral center in an area devoid of infected ticks as a source of endocarditis sera. PATIENTS: Sera from 30 consecutive patients with culture-proven subacute endocarditis between 1979 and 1981 were compared with 30 control sera collected between 1989 and 1990. In addition, sera from 20 consecutive patients with rheumatoid arthritis who were positive for rheumatoid factor were collected between 1991 and 1992. Sera were compared with a convenience sample from 15 patients who met the criteria for Lyme disease. MEASUREMENTS: Antibodies to B. burgdorferi were assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. IgM rheumatoid factor was quantified using solid-phase radioimmunoassay or latex agglutination techniques. RESULTS: Thirteen of 30 patients with endocarditis (43%) compared with 3 of 30 normal controls (10%) had increased levels of antibodies to B. burgdorferi (P < 0.01). Of these 13 patients, only 1 had an immunoblot consistent with previous infection. The others had nonspecific immunoblots: 5 showed isolated 60-kd reactivity; 1 patient had isolated 41-kd reactivity; and 6 had no bands of reactivity. Immunoblots of the 3 controls with increased antibodies showed only isolated 41-kd reactivity. Thus, the specificity of the B. burgdorferi antibody test in patients with endocarditis was only 60% (95% CI, 42% to 78%), compared with 90% (CI, 79% to 100%) in controls. No correlation was noted between IgM rheumatoid factor and antibodies to B. burgdorferi in patients with endocarditis (r = 0.2; P > 0.2). Only 1 of 20 patients with rheumatoid arthritis without known bacterial infections had antibodies to B. burgdorferi. CONCLUSIONS: Although a positive ELISA test for B. burgdorferi may be a "true positive," a positive serologic test alone does not ensure that the clinical problem is due to Lyme borreliosis. Cross-reactive antibodies to shared epitopes between B. burgdorferi and the endocarditis organism may account for the high false-positive results.

An acute form of T gamma lymphoproliferative disease presenting with massive splenomegaly--importance of immunophenotyping for diagnosis.

We describe a case of T gamma lymphoproliferative disease (T gamma LPD) which presented in an uncustomary acute onset in an adult with massive splenomegaly. Morphologically the cells represented monocytic leukemia. Karyotyping and equivocol special stain results suggested hairy cell leukemia. Gene rearrangement indicated a T lymphocytic malignancy. Immunocytochemistry stains were not definitive. Immunophenotyping by flow cytometry defined the cells as consistent with T gamma LPD (CD45+, CD56+, CD2+, CD3+, CD11b+, and CD38+; some cells CD8+; and CD57-). Although the cells did not have spontaneous activity, which is often the situation for most cases of T gamma LPD, the cells could be partially induced with exogenous interleukin 2 to exhibit in vitro cytotoxicity against a natural killer lymphocyte-susceptible target cell line (K562) but not a lymphocyte-activated killer target cell line (HEPG2). This report hopefully continues to increase the awareness of T gamma LPD as well as demonstrates an unusual acute form which could have been misdiagnosed unless a multidisciplinary approach, especially including flow cytometric immunophenotyping, was used to evaluate the patient.

Nonprotein antigens of Borrelia burgdorferi.

Preparative thin-layer chromatograms of chloroform-methanol extracts of Borrelia burgdorferi (B31) sonicates showed four fractions (Rf values of 0.84, 0.81, 0.66 and 0.61) that stained with iodine vapors, orcinol, or phospray, suggesting the presence of lipid-, carbohydrate-, and phosphorus-containing compounds. Sera from patients with Lyme disease showed IgM or IgG antibody reactivity to hydrophobic fractions, designated F1 and F2, in both early and late stages of the disease. Lack of constitutive amino acids in these fractions was shown by protein, amino acid, and peptide detection analyses. Sera from patients with syphilis, systemic lupus erythematosus, and antiphospholipid syndrome reacted to one or both of the fractions. Adsorption of sera from Lyme disease patients with intact B. burgdorferi resulted in significantly different pre- and postadsorption patterns of reactivity by whole cell ELISA, whereas adsorption with F1 and F2 resulted in similar pre- and postadsorption patterns. These fractions may not be present in aqueous whole cell or whole cell lysate ELISA antigens or in immunoblots.

Lyme borreliosis in Bell's palsy. Long Island Neuroborreliosis Collaborative Study Group.

Lyme borreliosis (LB) causes a range of neurologic manifestations, the most common of which is facial nerve paralysis. To evaluate nervous system LB, we organized a neurologic collaborative study group in Suffolk County, NY, a region of high LB incidence. Between July and September 1989, LB serologies were performed on all patients with new-onset Bell's palsy. Seven of 32 had serologic evidence of LB at onset. One, initially seronegative, was highly seropositive 5 weeks later. In the five in whom we examined CSF, there was no evidence of intrathecal synthesis of specific antibody. In highly endemic areas, LB may be responsible for 1/4 of cases of Bell's palsy. Rarely, the palsy may occur prior to the development of a measurable antibody response, indicating a need for follow-up serologic testing.

Description, analysis and impact of major solar activity during recent U.S. Shuttle missions.

Since STS-26, three large solar events have occurred during Shuttle missions; a geomagnetic storm during STS-29 and solar particle events (SPEs) during STS-28 and -34. The maximum dose to a crew attributed to an SPE was estimated to be 30 microGy (70 microSv). Time-resolved dosimetry measurements of the SPE dose during STS-28 were made using the Air Force Radiation Monitoring Equipment (RME)-III. Comparison of calculated and measured dose demonstrated a discrepancy, possibly a result of deficiencies in the geomagnetic cutoff model used. This experience demonstrates that dose from an SPE is strongly dependent on numerous factors such as orbit inclination, SPE start time, spectral parameters and geomagnetic field conditions; the exact combination of these factors is fortuitous. New sources of data and procedures are being investigated, including real-time tracking of auroral oval positions or determination of particle cutoff latitudes, for incorporation into operational Shuttle radiation support practices.

In vitro enhancement of natural cytotoxicity by atrial natriuretic peptide fragment 4-28.

The presence of atrial natriuretic peptide (ANP) binding sites in the thymic cortex, medulla, and splenic white pulp suggests that this peptide may have immunoregulatory activity. We examined the effect of ANP on human natural killer (NK) cell activity. ANP significantly augmented NK cell cytotoxicity after twenty-four hours of incubation but had no effect on NK activity after short-term incubations of one hour. In addition, atrial natriuretic peptide did not effect the expression of natural killer or T cell surface markers. This study demonstrates that atrial natriuretic fragment 4-28 enhances natural killer cell activity.