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Velocity sedimentation in a zonal rotor using gradients of uniform osmolarity was used to separate leukocyte subpopulations from human blood and bone marrow. The separations were performed at high sedimentation rates having the advantage of rapidity over conventional unit gravity separations. Myeloid stem cells (CFU-C) and cells reactive with phytohemagglutinin (PHA), Concanavalin A (Con A), and in mixed leukocyte culture (MLC) were separated and their sedimentation profiles obtained. CFU-C sedimented ahead of lymphoid cells and behind mature myeloid elements. Two distinct marrow subpopulations separated by velocity sedimentation were consistently stimulated by Con A, and variably stimulated by PHA and MLC reactions. Both large cells (predominantly myeloid) and small cells (predominantly lymphoid) from bone marrow were stimulated by Con A in [3H]thymidine incorporation assays. When separated subpopulations showing stimulation by Con A were mixed, inhibition of [3H]thymidine incorporation resulted.

Seventy-nine patients with metastatic or unresectable bronchogenic carcinoma were treated with a regimen of combination chemotherapy which included methotrexate, Oncovin (vincristine), cyclophosphamide, and adriamycin (MOCA), and were randomized to receive no additional therapy, immunotherapy with bacillus Calmette-Guérin, or immunotherapy with Corynebacterium parvum (C. parvum). The response rate and estimated median survival time were 68% and 42 weeks in small cell carcinoma and 18% and 29 weeks in other histologic types. Improved survival correlated with high performance status and response to therapy. Immunotherapy did not improve response, time to progression, or hematopoietic tolerance of chemotherapy. C. parvum was associated with significant morbidity and was poorly tolerated. MOCA appears to be of modest value in the treatment of bronchogenic carcinoma, particularly of the small cell type. A role for immunotherapy remains unproven.

Both murine and human bone marrow cells were cultured in plasma clots which were formed inside diffusion chambers implanted into cyclophosphamide- and saline-treated mice. After an initial fall, the number of mouse bone marrow cells and numbers of mouse myeloid stem cells (CFU-C) and agar cluster-forming units rose faster in the cyclophosphamide-treated animals. These hosts also favored formation of myeloid (CFU-D-G) and erythroid (CFR-D-E) colonies and myeloid higher than those of CFU-C from the same marrow population. These observations suggest the existence of humoral factors stimulating granulocyte progenitor cell replication and differentiation. At its best the increment of CFU-D-E number was equivalent to that caused by a single 0.1 unit erythropoietin dose. Culture of normal human marrow cells resulted in colonies in the plasma clot containing only granulocytes and macrophages. Cyclophosphamide-treated host animals were essential for human CFU-D-G development. Plating efficiency for human marrow myeloid colonies was better in the conventional in vitro agar cultures than in diffusion chambers.

The ability of syngeneic peripheral leucocytes to cure marrow aplasia was tested in a patient with paroxysmal nocturnal haemoglobinuria (P.N.H.). Transfusion of 7.1X10(10) white cells obtained by leucopheresis from an identical-twin donor, providing 3.4X10(4) myeloid progenitors (C.F.U.-C)/kg, failed to improve marrow function within two months. In contrast, transfusion of 1.3X10(10) nucleated bone-marrow cells, representing 6.4X10(4) C.F.U.-C/kg, from the same donor resulted in prompt bone-marrow recovery. These observations support the hypothesis that aplastic anaemia in P.N.H. is a stem-cell defect that may be corrected by the simple infusion of relatively small numbers of normal bone-marrow cells. They also seem to indicate a distinct advantage of marrow cells over peripheral-blood mononuclear cells in their ability to correct marrow aplasia.

Marrow cells of known HLA type were incubated with HLA antiserum plus complement and then plated in soft agar. Colony formation was consistently inhibited by appropriate HLA antisera. Mixing experiments excluded an indirect effect on CFU-C by lysis of mature leukocytes. We conclude that human CFU-C express HLA antigens.

Peripheral blood cells of 17 patients with myeloproliferative disorders were grown in vitro to form mixed colonies (CFU-mix). CFU-mix were found in four of nine patients with chronic phase chronic myelocytic leukaemia (CML) at levels 28- to 132-fold higher than the upper limits of normal subjects. These patients also had higher than normal levels of circulating granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. Development of blastic crisis was associated with disappearance of CFU-mix from the blood. CFU-mix was detected in the blood of three of five patients with polycythaemia vera (PV) and two patients with myelofibrosis with myeloid metaplasia (MMM) at levels slightly higher than those of normal subjects. Our results are consistent with a clonal expansion of multipotent haematopoietic precursor cells in CML and abnormal kinetics of this cell in CML, PV and MMM.

There are two genes related to the viral erbB gene in the human genome. c-erbB-1 is the same as the gene for the epithelial-growth-factor (EGF) receptor, and c-erbB-2 encodes a receptor-like protein very similar to, but distinct from, the EGF receptor. Hybridisation analysis of DNA from 101 fresh human malignant tumours showed that the c-erbB-2 gene was amplified in 5 of 63 adenocarcinomas and none of 38 other types of tumours, whereas the c-erbB-1/EGF-receptor gene was amplified only in 1 of 8 squamous-cell carcinomas. Thus, the protein products of the amplified c-erbB-2 gene may have a role in the evolution of adenocarcinomas, as does the EGF receptor in some squamous-cell carcinomas.

We have applied direct dideoxy sequencing of DNA fragments amplified in vitro by the polymerase chain reaction to the detection of mutations in exon 1 of the c-K-ras gene in human pancreatic adenocarcinomas. Four fresh frozen primary tumors, one metastatic tumor, and twelve formalin-fixed paraffin embedded tumors were analysed. Only three cases showed a possible mutation in codon 12 in a small population of cells, in contrast to the high frequency reported for this alteration with the RNAase A protection assay and allele-specific oligoxynucleotide hybridization. No major difference in sensitivity was found between DNA sequence analysis, and the latter method. Our results suggest that if c-K-ras mutations are indeed present in pancreatic adenocarcinomas at the high frequency reported by others, they must be confined to a small fraction of the cell population to escape detection by direct sequencing. Such a phenomenon would have implications for c-K-ras mutations in the pathogenesis of pancreatic adenocarcinomas.

Kinins are naturally occurring vasoactive polypeptides thought to be mediators of acute inflammatory responses. Kinins are released from a plasma protein substrate by glass-activated plasma enzymes (kallikreins) or by isolated intact granulocytes. Cortisol in concentrations of 2.5 x 10(-6) to 2.5 x 10(-5)M prevented the release of active kinin from substrate by granulocytes or contact with glass. Deoxycorticosterone, progesterone, and etiocholanolone in comparable concentrations were significantly less effective in preventing kinin release. Plasma obtained from patients receiving prednisone released no kinin after activation by glass and less kinin than control plasma when exposed to granulocytes. Cortisol also partially inhibited the release of kinin by purified urinary kallikrein. Certain adrenocorticosteroids may exert their anti-inflammatory effect by inhibiting the release of plasma kinins. Steroids may act in part by preventing interaction between the activated kallikrein and its substrate.

The mechanism by which macrophages kill facultative intracellular parasites is not known. A test system is described utilizing human macrophages derived from blood monocytes and the opportunistic pathogen Listeria monocytogenes. In this system, bacterial killing was impaired under hypoxic conditions. Under aerobic conditions, addition of Clofazimine (B663), a phenazine derivative effective in the treatment of human leprosy, potentiated the killing of Listeria by macrophages. Potentiation occurred at drug concentrations usually attainable in man and which are not directly injurious to the bacteria. The enhanced bactericidal activity occurred only in the presence of oxygen; the drug induced increased leukocyte oxygen consumption which was insensitive to cyanide.

Human alveolar macrophages were lavaged from surgically resected lungs and from lungs of normal subjects. Macrophages that had been purified by glass adherence were maintained in tissue culture for as long as 54 days. After 3-4 wk in vitro they underwent transformation into multinucleated giant cells. These aged cells had more than 30 times the phagocytic capacity that the same group of cells had had after 1 day in vitro. Phagocytosis of heat-killed Candida albicans was inhibited by iodoacetate, sodium fluoride, potassium cyanide, and low partial pressures of oxygen, suggesting that these cells require both oxidative and glycolytic energy sources for maximal particle ingestion. Alveolar macrophages and monocyte-derived macrophages killed Listeria monocytogenes with similar efficiency, but neutrophils were more efficient than either of the other cell types. Bacterial killing is probably not dependent upon myeloperoxidase in the monocyte-derived macrophage or in the alveolar macrophage since histochemical stains for peroxidase do not stain either cell type. C. albicans blastospores, which are killed by neutrophils and monocytes that contain myeloperoxidase, were not killed by human alveolar macrophages during the 4 hr of observation. Large cells with supernormal phagocytic capacity were recovered from patients with postobstructive pheumonia and from one patient with recurrent bacterial pneumonia, indicating that macrophage function can be altered in certain disease states. Human alveolar macrophages are unique human phagocytes in their dependence on an oxygen tension greater than 25 mm HG for maximal phagocytosis. Carbon dioxide tensions as high as 70 mm Hg did not alter phagocytosis when the pH of the medium was held constant. These data suggest that the increased susceptibility to pneumonia of patients with chronic bronchitis or atelectasis may be in part related to suboptimal phagocytosis by macrophages in areas of the lung with depressed oxygen tension.

In studies of 13 normal adults to determine the blood cell types responsible for interferon production induced by phytohemagglutinin, the following observations were made. (a) In cultures containing 96-100% pure macrophages derived from blood monocytes, no interferon was detected in either the presence or the absence of phytohemagglutinin for up to 92 hr. (b) In cultures of 99.5-100% pure lymphocytes, low levels of interferon were detected in the presence, but not in the absence, of phytohemagglutinin. (c) An average fivefold increase in interferon titers occurred when pure lymphocytes were combined with the macrophages in culture with phytohemagglutinin. The peak response of interferon occurred at 68 hr after the initiation of the combined cultures. For maximum response, phytohemagglutinin was required for the duration of the culture, and both cell types in association were necessary. Medium from phytohemagglutinin-stimulated macrophages or lymphocytes could not substitute for the corresponding intact cell. However, frozen-thawed macrophages in combination with lymphocytes and phytohemagglutinin produced an intermediate interferon response. An increase in either cell type produced an increased response in the range studied: lymphocytes, 0.45-1.8 x 10(6) per ml; and macrophages, 0.5-2.1 x 10(5) per ml. Syngeneic fibroblasts, HeLa cells, or mouse macrophages could not substitute for the human macrophages in the combined cultures with phytohemagglutinin. (d) Although all cultures producing interferon showed some degree of transformation (thymidine-(3)H incorporation into deoxyribonucleic acid), no direct correlation between the degree of phytohemagglutinin-induced lymphocyte transformation and the interferon titers was observed.The demonstration of macrophage-lymphocyte interaction in the production of interferon is of interest in view of the known interrelationship of these same cell types in antibody synthesis and cellular immunity.

Bradykinin is a potent constrictor of the human umbilical artery and vein and the ductus arteriosus of the lamb in vitro at oxygen tensions above 40 mm Hg (comparable to those in the newborn infant). Bradykinin is also capable of producing remarkable dilatation of the pulmonary vasculature of the lamb. Theoretically, kinins are capable of effecting some of the rapid circulatory changes required of the neonate. The present study was undertaken to investigate the role of kinins as mediators of such changes. The concentration of bradykinin in the cord blood of 56 newborn infants at the time of birth was significantly higher than the blood level in adult subjects (12.8 +/- 4.3 ng/ml compared with 2.0 ng/ml or less). Cord arterial blood contained inactive kinin precursor (kininogen) and inactive kinin-releasing enzyme (kallikrein). Plasma kallikrein was activated, with subsequent kinin formation and kininogen depletion, by exposure to neonatal granulocytes or by a decrease in the temperature of cord blood from 37 to 27 degrees C. A comparable decrease in the temperature of umbilical arterial blood occurs at the time of birth. Activation of kallikrein by neonatal granulocytes was dependent on cell concentration and required oxygen tensions comparable to those in the neonate but above the range in the fetus. Granulocytes of the neonate, unlike those of adult subjects, lacked kininase activity.Thus, bradykinin can constrict and dilate vessels as required for the transition of fetal to neonatal circulation. Bradykinin can be produced in plasma of the newborn by decreases in temperature, such as occur in the umbilical blood at birth, and by exposure to granulocytes which are present in the circulation in increased numbers shortly after birth. We propose that bradykinin is produced at birth and may be a mediator of neonatal circulatory changes.

These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California Medical Center, San Francisco. Taken from transcriptions, they are prepared by Drs. Martin J. Cline and Hibbard E. Williams, Assistant Professors of Medicine, under the direction of Dr. Lloyd H. Smith, Jr., Professor of Medicine and Chairman of the Department of Medicine.

Monocytes isolated from the peripheral blood of tuberculin-positive and tuberculin-negative donors were exposed to PPD, extensively washed, and incubated with autologous or homologous lymphocytes. Lymphocyte transformation was measured morphologically and by incorporation of (14)C-labeled thymidine. Monocytes from tuberculin-positive subjects induced transformation of autologous lymphocytes in 19 of 29 experiments. Studies to define the optimal conditions of exposure to monocytes to PPD and to autologous lymphocytes showed that viable, metabolically intact monocytes are required. A ratio of only 1 monocyte to 100 lymphocytes sufficed to induce transformation; neutrophils were inactive. In general, PPD-sensitized monocytes failed to induce transformation of homologous lymphocytes from either tuberculin-positive or tuberculin-negative subjects. Direct contact between monocytes and lymphocytes was required for consistent transformation, and islands of transforming lymphocytes were observed around a central core of monocytes.

D-Amino acid oxidase has been identified within the granule fraction of human neutrophilic leukocytes. Leukocyte homogenates and purified kidney D-amino acid oxidase can utilize either isolated D-amino acids or some species of bacteria as substrates for the generation of hydrogen peroxide. When linked to leukocyte myeloperoxidase in vitro, purified D-amino acid oxidase constitutes a system lethal for certain bacteria. It is proposed that leukocyte D-amino acid oxidase and myeloperoxidase constitute a biochemically specific system for the recognition and killing of certain microorganisms.

Bone marrow colony formation in soft gel culture may be stimulated by substances elaborated by human peripheral blood leukocytes. In order to determine the cell type responsible for colony stimulation, peripheral leukocytes were separated by Ficoll-Hypaque gradients and differential glass adhesion. Morphologic, histochemical, and functional criteria were applied to determine the purity of the monocyte, lymphocyte, and neutrophil fractions. Using these cells as feeder layers and as a source of conditioned medium, evidence was obtained that the monocyte is the colony-stimulating cell of human peripheral blood. Activity greater than that of mixed white cells was obtained with monocyte underlayers, and only monocyte- and macrophage-conditioned media were shown to have significant colony-stimulating activity.

The neutrophils and monocytes of a patient with disseminated candidiasis were found to lack detectable levels of the lysosomal enzyme myeloperoxidase (MPO), although they had normal levels of other granule-associated enzymes. Leukocytes from one of the patient's sisters also lacked detectable MPO; leukocytes from his four sons contained approximately one-third of mean normal peroxidase levels. Neither the patient nor his affected relatives had experienced frequent or unusual bacterial infections. The phagocytic activity of the patient's MPO-deficient neutrophils was intact, and the cells displayed normal morphologic and metabolic responses to phagocytosis. In contrast to normal leukocytes which killed 30.5+/-7.3% of ingested Candida albicans in 1 hr, however, the patient's neutrophils killed virtually none. His leukocytes also failed to kill the strain of C. albicans recovered from his lesions, as well as other Candida species. These MPO-deficient neutrophils killed Serratia marcescens and Staphylococens aureus 502A at an abnormally slow rate, requiring 3-4 hr to achieve the bactericidal effect attained by normal leukocytes after 45 min. No other abnormalities in his cellular or humoral immune responses were demonstrated. These findings suggest that hereditary MPO deficiency is transmitted as an autosomal recessive characteristic, that the homozygous state conveys enhanced susceptibility to disseminated candidiasis, and that MPO is necessary for candidacidal activity in human neutrophils. Although lending support to the suggested bactericidal role of MPO in leukocytes, the data indicate that alternative bactericidal mechanisms, effective in the absence of MPO, are functionally dominant in the human neutrophil.

A quantitative assay of candidacidal activity based on differential staining of non-viable Candida albicans by methylene blue was developed and applied to studies of leukocytes from normal individuals and patients with fungal and other infections. Serum factors were necessary for optimal phagocytosis of C. albicans but lacked direct candidacidal activity. Normal human neutrophils (38 studies) killed 29.0 +/- 7.4% of ingested C. albicans in 1 hr. Eosinophils and monocytes killed a smaller percentage. Neutrophil candidacidal activity did not require protein or ribonucleic acid synthesis by the leukocyte but was inhibited by anaerobic conditions, potassium cyanide, and colchicine. Leukocytes of a patient with hereditary myeloperoxidase deficiency and of three children with chronic granulomatous disease phagocytized C. albicans normally, yet failed to kill them. Our data suggest that the neutrophil can play an important role in resistance to Candida infection and that the lysosomal enzyme myeloperoxidase and its oxidant substrate hydrogen peroxide are the major participants in neutrophil candidacidal activity.

These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California Medical Center, San Francisco. Taken from transcriptions, they are prepared by Drs. Martin J. Cline and Hibbard E. Williams, Associate Professors of Medicine, under the direction of Dr. Lloyd H. Smith, Jr., Professor of Medicine and Chairman of the Department of Medicine.

These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California Medical Center, San Francisco. Taken from transcriptions, they are prepared by Drs. Martin J. Cline and Hibbard E. Williams, Assistant Professors of Medicine, under the direction of Dr. Lloyd H. Smith, Jr., Professor of Medicine and Chairman of the Department of Medicine.