Calculations on crystal packing of a flexible molecule, Leu-enkephalin.

Crystal packing calculations have been carried out on a substantial number of conformations of Leu-enkephalin; namely, those obtained both from crystal structures and from energy minimizations on isolated molecules, and with and without waters of crystallization. The known crystal structures represent the most energetically stable packings found. The conformations of the enkephalin molecules in the crystal are not the most stable for an isolated molecule; i.e. intermolecular interactions force the isolated molecule to change conformation in order to achieve a small packing volume and an optimal packing energy in the crystal. It is found that the packing energy of an enkephalin molecule is a reasonably smooth function of its molecular volume in the unit cell, if structures with intermolecular hydrogen bonding are excluded, and is substantially independent of other details of the molecular conformation or of the crystal packing. Hydrogen bonding provides additional stabilization of the crystal structure, and would likely permit crystallization of the system if it is sufficiently dense. Solvent molecules further stabilize the structure when they can also provide intermolecular hydrogen bonds.

Effect of cryopreservation on purging of leukemic marrow with alkyl-lysophospholipids.

Alkyl-lysophospholipids (ALP) are reported to be selectively cytotoxic to neoplastic cells and have been demonstrated to be an effective purging agent for autologous marrow transplantation in a murine model, WEHI-3B. We studied the effect of in vitro treatment of normal bone marrow cells with ALP and of cryopreservation on spleen colony-forming units (CFU-s) in irradiated, syngeneic Balb/c mice. We found that exposure of cells to doses of ALP of 5 or 20 micrograms/ml followed by cryopreservation had no effect on CFU-s, but that a dose of 100 micrograms/ml was toxic. A simulated remission marrow was prepared by mixing normal murine bone marrow cells with WEHI-3B myelomonocytic leukemic cells to give a 1%-2% concentration of leukemic cells. These were exposed to 0- to 100-micrograms/ml concentrations of ALP for 24 h, cryopreserved, thawed, and injected into the tail veins of lethally irradiated syngeneic mice, and survival measured. It was found that concentrations of ALP from 10 to 50 micrograms/ml resulted in long-term disease-free survivors. These results established the fact that cryopreservation did not alter the effectiveness of ALP in purging marrows of residual leukemic cells.

Response of human multiple myeloma-derived cell lines to alkyl-lysophospholipid.

Clinical studies using high-dose chemotherapy and autologous bone marrow transplantation in multiple myeloma report improvement in tumor regression. The potential success of this approach may be compromised because of the myeloma cell burden. A promising approach is chemopurging of bone marrow with alkyl-lysophospholipids (ALP), a new class of antitumor agents. Using a limiting dilution assay, we tested ALP against several drug-resistant human myeloma cell lines: RPMI 8226/S, 8226/DOX40, 8226/LR-5, 8226/MR-20, 8226/DOX1V, and 8226/MDR10V. IC50 values were approximately 6 microg/mL, for all cell lines evaluated. ALP was effective against all cells, with mean log kills ranging from 2.48 to 4.95. Cytotoxicity was temperature-dependent. Survival curves of cell lines exposed to ALP showed a steep slope during the first 4 hours of exposure, with little additional cell kill after 24 hours. Dose-response curves after drug exposure for 4 and 24 hours reached plateaus at 75 and 25 microg/mL, respectively. Increasing concentrations of ALP caused a sustained increase in resting [Ca2+]i of sufficient magnitude to induce cell death. These results suggest that ALP is an effective cytotoxic agent even in cells known to be resistant to alkylating agents and other classes of cytotoxic drugs.

A comprehensive study of umbilical cord blood cell developmental changes and reference ranges by gestation, gender and mode of delivery.

OBJECTIVE: The purpose of this study was to determine the normal hematological values in cord blood during gestation, the impact of the type of delivery and differences in gender. STUDY DESIGN: The database included 10 287 live births of 30-44 weeks gestation from cesarean or vaginal deliveries. Cord blood was collected into bags containing lyophilized heparin. Specimens were stored for 24 h or less and analyzed using the SysmexXE-2100. Data from cesarean births were used to evaluate developmental hematopoietic changes. RESULT: Increases during maturation occurred in hemoglobin, hematocrit, red blood cell count, and decreases in mean corpuscular volume and mean corpuscular hemoglobin. The number of nucleated red blood cells per 100 white blood cells decreased but absolute counts remained constant. Quantitative counts of white blood cells, neutrophils, monocytes (MON), eosinophils and lymphocytes (LYMP) increased, but percentages of lymphocytes and monocytes decreased. Platelets increased from 30-35 weeks. CONCLUSION: Reference ranges were established for cord blood. Erythroid and myeloid cells show developmental changes. Mode of delivery has a significant effect on hematologic values. Only a rare parameter showed differences based on gender. The cord blood complete blood cell count has the potential for providing relevant clinical information for managing neonatal patients.

The in vivo development of plasma cells: a morphologic study of human cerebrospinal fluid.

This is a case study of a patient with the clinical diagnosis of meningoencephalitis. It demonstrates the in vivo development of plasma cells in the central nervous system. All the stages of in vitro mitogen-induced lymphocyte transformation previously described by light and electron microscopy are recapitulated in this analysis of cerebrospinal fluid. It is postulated that this represents a local humoral immune reaction in the central nervous system and provides morphologic support for the concept that the gamma globulin in cerebrospinal fluid is produced locally in part, in addition to its origin from the plasma proteins.

Basophilic meningitis secondary to lymphoma.

In this case report of basophilic meningitis, the patient was a 5-year-old girl under treatment for a diffuse lymphocytic lymphoma. She presented with headache and bilateral papilledema. Cerebrospinal fluid examination showed 60 percent basophils. Subsequent specimens showed a rare blast. It is postulated that after the lymphoma cells had spread to the meninges, a cell-mediated immune reaction was initiated with the appearance of basophils in the exudate.

New ultrastructural observations: parallel tubular arrays in human T gamma lymphoid cells.

T gamma cells are E-rosetting cells bearing Fc receptors for IgG (E+, Fc gamma + cells). Third population (non-T, non-B) lymphoid cells are also Fc gamma + cells and contain unique inclusions called parallel tubular arrays (PTA). Although T gamma cells and third population lymphoid cells should belong to a similar population of cells, previous ultrastructural studies on purified T gamma cells have failed to reveal the presence of PTA. In this study, we have unequivocally demonstrated PTA in the majority of T gamma cells using simple rosetting techniques. A total of 76 EA hu-rosettes and 108 EA ox-rosettes prepared from an E+ enriched fraction (using sheep erythrocytes as marker particles) were directly examined by electron microscopy. PTA were found in 87% of the EA hu-rosettes and 82% of the EA ox-rosettes. Ammonium chloride, commonly used in other laboratories to lyse erythrocytes during the purification procedure was found to cause a marked decrease in the number of ultrastructurally distinct PTA profiles. In contrast, hypotonic lysis had no effect on cellular ultrastructure. This study showed for the first time that T gamma cells are ultrastructurally similar to other Fc gamma + lymphoid cells and contain PTA as a distinct marker. The significance of our findings to the basic function of this E+ Fc gamma + lymphoid population is discussed.

Arm exercise testing with myocardial scintigraphy in asymptomatic patients with peripheral vascular disease.

Arm exercise with myocardial scintigraphy and oxygen consumption determinations was performed by 33 men with peripheral vascular disease, 40 to 74 years of age (group 2). None had evidence of coronary disease. Nineteen age-matched male control subjects (group 1) were also tested to determine the normal endurance and oxygen consumption during arm exercise in their age group and to compare the results with those obtained during a standard treadmill performance. The maximal heart rate, systolic blood pressure, pressure rate product, and oxygen consumption were all significantly lower for arm than for leg exercise. However, there was good correlation between all these parameters for both types of exertion. The maximal heart rate, work load and oxygen consumption were greater for group 1 subjects than in patients with peripheral vascular disease despite similar activity status. None of the group 1 subjects had abnormal arm exercise ECGs, while six members of group 2 had ST segment changes. Thallium-201 scintigraphy performed in the latter group demonstrated perfusion defects in 25 patients. After nine to 29 months of follow-up, three patients who had abnormal tests developed angina and one of them required coronary bypass surgery. Arm exercise with myocardial scintigraphy may be an effective method of detecting occult ischemia in patients with peripheral vascular disease. Those with good exercise tolerance and no electrocardiographic changes or 201T1 defects are probably at lower risk for the development of cardiac complications, while those who develop abnormalities at low exercise levels may be candidates for invasive studies.

Leukemic cells as probes for sequential functional differentiation of the human granulocyte.

The purpose of this study was to sequence the functional properties of the neutrophil during maturation, using leukemic cells as experimental probes. Twenty-nine cases of acute leukemia, derived from granulocytic precursors, were studied. The functional tests that were evaluated included phagocytosis, bactericidal activity, random locomotion, and chemotaxis. These functional properties were correlated with Fc receptors and the stimulated nitroblue tetrazolium dye reduction test. The results indicated that the various functions of the neutrophil are acquired sequentially rather than concurrently. It is postulated that the sequence of functional differentiation is: phagocytosis leads to microbial killing leads to random locomotion leads to chemotaxis.

Functional differentiation in acute monoblastic leukemia.

Blasts from the bone marrow of a patient who had acute monoblastic leukemia were studied for functional maturity. Classification of the leukemia was based on cytochemical stains. The blasts were negative when stained with Sudan black B and did not have specific esterase activity. They were rich in alpha-naphthyl butyrate esterase, which was inhibited by fluoride. Functional assays included phagocytosis of Candida albicans, fungicidal activity measured by differential Giemsa staining of the ingested C. albicans, and locomotion using the agarose method to evaluate both random migration and chemotaxis. At a ratio of 20 yeast cells to one monoblast, 80% of the blasts could be stimulated to phagocytize an average of 4.4 organisms. These results compared favorably with the phagocytic potential of normal human monocytes. Candidacidal activity was present, but reduced. At high ratios of yeasts to monoblasts, only ten organisms were killed for every 100 phagocytic blasts. This correlates with the absence of myeloperoxidase activity demonstrated by negative Sudan black B staining. Neither chemotaxis nor random migration could be demonstrated, indicating that monoblasts lacked the apparatus necessary for locomotion. Extrapolation of these findings to normal monocyte maturation suggests that phagocytosis is acquired prior to microbicidal activity, which develops prior to locomotion.

The effect of various cell separation procedures on assays of neutrophil function. A critical appraisal.

Neutrophils are considered fragile cells, easily damaged by improper handling. Assays of neutrophil function frequently require preliminary isolation procedures that may be potentially harmful. The authors did an extensive investigation of the effects of currently used isolation procedures on a broad spectrum of functional assays that included: 1) phagocytosis; 2) bacterial killing by a culture technique and tritiated thymidine labeling; 3) candidacidal activity by methylene blue dye exclusion and differential Giemsa staining; 4) quantitative unstimulated and histochemical stimulated nitroblue tetrazolium dye reduction; 5) chemiluminescence; 6) random locomotion; and 7) chemotaxis by the agarose method. In addition, cells were examined morphologically by electron microscopy, and the granule density was quantitated by morphometric analysis. Isolation techniques included erythrocyte sedimentation, hypotonic lysis, gradient density separation using Ficoll-Hypaque, and counterflow centrifugal elutriation. The purest neutrophil suspensions were obtained by density gradient separation and counterflow centrifugal elutriation with mean neutrophil percentages of greater than or equal to 94%. Regardless of the isolation procedure, neutrophils were similar in all groups (P greater than .05) in the following studies: phagocytosis, nitroblue tetrazolium dye reduction, bacterial killing, candidal killing, inhibition of candidal germ tube formation, and cytoplasmic granulation. Differences were noted in assays of neutrophil migration and chemiluminescence. Neutrophil suspensions isolated by counterflow centrifugal elutriation and Ficoll-Hypaque had the highest scores for random migration and chemotaxis. These differences can be related to the purity of the neutrophil suspension rather than the harmful effects of the isolation procedures. Erythrocyte contamination affected both the slope and the time to peak response in the chemiluminescence assay. Exposure of neutrophils to cold temperatures (0-4 degrees C) for 1.5 hours impaired both random locomotion and chemotaxis. Current recommendations for the storage, transport, and preparation of leukocytes for neutrophil function studies need to be reassessed.

Neutrophil migration under agarose: quantitation and variables.

Several variables have been evaluated for the in-vitro measurement of neutrophil migration under agarose. Commonly used anticoagulants do not alter migration. Cells may be collected in heparin, sodium citrate, or EDTA. Neither hypotonic or ammonium chloride lysis of erythrocytes affects chemotactic activity. Saponin, however, decreased both leukocytic viability and migration. Passage of neutrophils through a Hypaque-Ficoll density gradient improved migration scores, presumably by removing contaminating material and cells. Both lymphocyte and erythrocyte contamination decreased test scores. Platelets did not have a detrimental effect on neutrophil migration. Fresh serum was the most potent chemotactic agent. This was followed, in order, by zymosan-activated serum, N-formyl-L-methionyl-L-phenylalanine, casein, and Escherichia coli supernatant. A scoring method that combines features of both counting leukocytes and measuring the migration distance has been devised. The technic has a coefficient of variation of 9.2%. Scores for the normal adult population show a gaussian distribution.

Programmed cell death of the normal human neutrophil: an in vitro model of senescence.

The present study provides experimental data which indicate that the neutrophil is ideal for studying programmed cell death or apoptosis in vitro. Neutrophils can be obtained from human peripheral blood in large numbers with minimal experimental manipulation and are easily separated from other leukocytes, providing nearly pure cell suspensions. The neutrophil life span in vitro is sufficiently short to allow observations to be made within eight hours after experimental manipulation. Neutrophils can also be easily maintained in serum-free, chemically defined media which can be systematically altered, thereby defining specific variables that influence the apoptotic process. Since the neutrophils do not need an exogenous trigger to undergo programmed cell death, it is also an excellent model to study senescence. It was determined from this study that neutrophils undergo apoptosis most efficiently at 37 degrees C, a temperature requirement for physiologic cell death. Neutrophils undergo apoptosis at a slightly faster rate and maintain membrane integrity better when incubated in a tissue culture medium (e.g., RPMI 1640) compared with a balanced salt solution (e.g., HBBB). Cycloheximide, an inhibitor of protein synthesis, was shown to accelerate apoptosis in a dose-dependent manner. The presence of Zn++ significantly decreased the rate of apoptosis, whereas the presence of Ca++ and Mg++ had no apparent effect. These studies indicate that the process of senescence, culminating in cell death, is subject to modulation by a variety of agents and experimental conditions. In addition, the ultrastructural features of neutrophils undergoing programmed cell death in vitro were compared in detail to those occurring in vivo and were found to be comparable.

A Philadelphia chromosome positive CML patient with a unique translocation studied via GTG-banding and fluorescence in situ hybridization.

The Philadelphia (Ph) chromosome was the first consistently occurring chromosome abnormality associated with a single cancer type, chronic myelogenous leukemia (CML). This translocation has since been reported with other chromosome abnormalities. The present report describes a case of Ph chromosome positive CML with a unique complex translocation identified using molecular cytogenetics in addition to routine techniques. GTG-banding revealed abnormalities in at least chromosomes 9, 13, 17, and 22. Fluorescence in situ hybridization (FISH) studies were performed as an adjunct to conventional cytogenetic analyses. Using FISH with the Oncor bcr/abl probe, the Ph translocation previously hypothesized was confirmed. Applying FISH with paired painting probes in various combinations, a complex translocation involving chromosomes 9, 13, 15, 17, and 22 was observed. The results of the GTG-banding and FISH studies were compared with each other and correlated with those of the hematological findings. In an extensive search of the medical literature database (Medline, Health, Cancerlit, Ovid, and CINAHL) spanning nearly three decades (1965-1994), we found no previous report of this specific translocation. Therefore, to the best of our knowledge, this is a unique translocation associated with Ph chromosome positive CML.

Experimental studies on the role of alkyl lysophospholipids in autologous bone marrow transplantation.

The selective cytocidal effect of alkyl lysophospholipids against neoplastic cells while sparing normal cells make these ideal candidates for purging leukemic cells from bone marrows obtained during remission. To test the feasibility of such an approach, a murine model and an in vitro human cell model were developed. In the murine system a mixture of normal bone marrow cells and WEHI IIIB myelomonocytic leukemic cells was incubated with varying doses of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-Me) for 24 hr before being injected into tail veins of lethally irradiated Balb/c mice. At doses of 20 and 100 micrograms/ml, long-term survivors were noted. The additional steps of freezing and thawing following incubation resulted in significantly longer survival with doses of 10 to 50 micrograms/ml, but were toxic to marrow stem cells at 100 micrograms/ml. In the in vitro model, normal marrow progenitor cells and leukemic cells (the promyelocytic cell line HL60) were exposed to varying concentrations of ET-Me for 1 and 4 hr alone or mixed, and clonogenicity was assayed by colony formation in semisolid medium during 7-14 days' incubation. At doses up to 100 micrograms/ml exposed for 4 hr normal progenitor cells were spared and HL60 colonies eliminated. Other phospholipids analogues were less effective in eliminating leukemic cells, but spared normal progenitor cells. A survey of fresh leukemic cells found varying degrees of sensitivity to ET-Me, indicating the need for testing a variety of compounds. These studies clearly indicated the potential usefulness of alkyl lysophospholipid compounds in selectively purging leukemic cells from remission marrows for autologous bone marrow transplantation.

Effects of isokinetic training on the rate of movement during ambulation in hemiparetic patients.

The purpose of this study was to determine the effects of isokinetic training on the rate of movement during ambulation in hemiparetic patients. Ten male and 10 female subjects, aged 40 to 75 years, participated in the study. The 20 hemiparetic subjects were assigned randomly to either a control group or an experimental group. All of the subjects participated in a conventional therapeutic exercise program and gait training. The experimental group also received isokinetic training on the Kinetron exercise machine as part of their program. Functional ambulation profile tests were administered to each subject before and after the five-week experimental period. All of the subjects showed improvement in the rate of ambulation and in overall ambulation performance. The differences in ambulation times and functional ambulation profile scores between the two groups were shown to be insignificant.

Ultrastructural morphometry in the diagnosis of Sézary syndrome.

Blood specimens from 87 patients and control subjects were prepared for electron microscopy and subjected to ultrastructural morphometric evaluation by using a computerized planimeter. A statistical comparison of means indicated that patients with Sézary syndrome could be distinguished from normal subjects and patients with reactive lymphocytosis, by using mean nuclear perimeter and form factor values. The lymphocytic nuclei from patients with infectious mononucleosis were more lobated on visual inspection than those from normal subjects; the difference in mean form factor values was statistically significant. The simple histogram method was most discriminatory and distinguished patients with Sézary syndrome from patients with other types of lymphoid leukemias and reactive lymphocytosis, including infectious mononucleosis. The histogram method could not, however, distinguish patients with Sézary syndrome from patients with T-cell chronic lymphocytic leukemia and Japanese T-cell leukemia. The use of bivariate graphic displays (plotting nuclear size and shape measurements) placed the lymphoid cells of the various types of lymphoproliferative disorders into distinct morphometric domains. Computerized morphometric techniques may, therefore, be of greater value when the range of possible diagnoses is large.

Binding isotherms by continuous-flow dynamic dialysis.

The classical dynamic dialysis technique for the determination of a protein-ligand binding isotherm has been modified by the introduction of a flow cell in which the dialysate on the sink side of the membrane is continuously eluted with a constant flow of eluting buffer and its ligand concentration measured. This new experimental method is termed continuous-flow dynamic dialysis (CFDD). A transfer function procedure for extracting the binding isotherm from the dialysis data is described. This is a more general technique (requiring only a verifiable assumption of linearity) than that previously used, in which the system was modelled using Fick's first law and which relied on the establishment of quasi-steady state conditions across the membrane. The present analysis uses the Laplace transform to effect deconvolution of the impulse response function of the cell from the dialysis data and, using a Fourier series approach, directly yields numerical data representing the free ligand concentration in equilibrium with the protein-ligand complex. The protein-ligand binding isotherm is obtained in parametric form, with time as the parameter.

Thoracic gallium uptake in patients with lymphomatoid granulomatosis.

Lymphomatoid granulomatosis (LG) is a rare condition with histological similarities to Wegener's granulomatosis and malignant lymphoma. Characteristically there is an angiocentric, angiodestructive lymphoreticular cell infiltrate. The lungs are usually affected, and, less frequently, the skin, nervous system, kidney, and bowel are involved. The prognosis is poor and frank lymphoma develops, in some cases terminally. The usual radiological appearance of the lungs consists of bilateral nodular lower zone opacities. The authors report two patients (siblings) with LG, and their gallium scans are presented. In each case there was a significant accumulation of gallium in the lungs at times of clinically active disease. The limited role of gallium imaging in this disease is discussed.