Hugh Ross's curious lymphocyte experiments

It is now almost 100 years since Hugh Campbell Ross began his experiments on white blood cells and cancer. By suspending peripheral blood cells in a solution of agar gel, he was able to observe changes in them provoked by various natural and artificial substances, which he named auxetics, kinetics, or augmentors, depending on the effects they produced. After his early experience he focused his attention particularly on lymphocytes in peripheral blood samples; he claimed to have observed rapid division in them, although at that time they were looked upon as end cells without a future. He contended that his results challenged what had become the orthodox view of the role of the nucleus in mitosis, asserting that cytologists had been led astray by relying on examination of fixed, dead structures. When doubt was cast on his claim to have induced cell division, his brother Ronald, Nobel laureate in physiology or medicine, came to his defense, but to no avail, and although he continued his experiments for a number of years they were quietly forgotten. Fifty years later the occurrence of mitotic division of peripheral blood lymphocytes was established beyond any doubt by Nowell and Hirschhorn.     

A personal view from a long-lasting collaborator on the research strategies of Marshall Nirenberg

In this review, I summarized transition in Dr. Marshall Nirenberg’s research interests during 1970s, from a view of a long-lasting collaborator. Nirenberg switched his research filed to neurobiology after his success in deciphering genetic code and being honored with the Nobel Prize in Physiology or Medicine in 1968. His targets were to obtain genetically pure population of neurons, i.e. neuroblastoma clones, to make somatic hydrid cells, to culture neuronal and muscle cells, and to produce monoclonal antibodies against whole retinal or neuroblastoma cells. He studied neurotransmitters, receptors, cyclic nucleotides, cell differentiation, secretion, synapse formation, and chemical recognition. Especially he liked his hypothesis for opiate tolerance and dependency as a model of cellular memory. Through these studies, he seemed to devote all his time of about 50 years from 1960s to decoding brain memory processes.     

In vivo kinetics of transduced cells in peripheral T cell-directed gene therapy: role of CD8+ cells in improved immunological function in an adenosine deaminase (ADA)-SCID patient.

We previously reported successful peripheral T cell-directed gene therapy in a boy with adenosine deaminase (ADA)-SCID. In the present study, to better understand the reconstitutive effect of this gene therapy on his immunological system, we investigated the in vivo kinetics and functional subsets of T cells in PBL. Apparent immunological improvements were obtained after infusion of transduced cells at more than 4 x 108 cells/kg/therapy/3 mo. Frequency of ADAcDNA-integrated cells in PBL, ADA activity in PBL and clinical improvement showed good correlation, even though CD8+ cells gradually became predominant in PBL. On the basis that polyethylene glycol (PEG)-ADA was maintained at the same dosage as before gene therapy, we consider that his immunological improvement resulted from the gene therapy itself. Most CD3+ cells in PBL after gene therapy expressed TCRalphabeta. Analysis of TCR repertoire based on TCR V region usage revealed no expansion of limited clones in his PBL. The T cell subset cells CD8+CDw60+ and CD8+CD27+CD45RA-, which are reported to provide substantial help to B cells, were maintained throughout the gene therapy. Furthermore, his reconstituted peripheral T cells helped normal B cells to produce substantial IgG in vitro. Expression of both Th1- and Th2-type cytokine genes was induced in his reconstituted T cells at the same comparably high level as in normal subjects. Collectively, these results provide evidence of persistent and distinct functions of transduced cells in this patient's PBL after gene therapy.     

Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of constriction of the required strains

A 'toxicity' test protocol is described here to be used for determining the bactericidal effect of the chemicals which are tested for their mutagenic activity by the Ames method. Two sets of strains, isogenic with the Ames tester strains except for their his character, are constructed. One set is the his+ derivatives of the tester strains which are used for measuring the survival of the inoculum cells after exposure to the chemical. The other set is the stable his- derivatives of the tester strains which are used for simulating the background growth in the Ames mutagenicity plate test. The per cent survival of the his+ cells in the inoculum in the presence of the 'filler cells' is used as a measure of the toxic effect of the chemical.     

Uneven distributions of naïve and memory T cells in the CD4 and CD8 T-cell populations derived from a single stem cell in an atomic bomb survivor: implications for the origins of the memory T-cell pools in adulthood

The processes that lead to the establishment and maintenance of memory T-cell pools in humans are not well understood. In this study, we examined the emergence of na?ve and memory T cells in an adult male who was exposed to an atomic bomb radiation dose of approximately 2 Gy in 1945 at the age of 17. The analysis presented here was made possible by our earlier observation that this particular individual carries a hematopoietic stem cell mutation at the hypoxanthine phosphoribosyltransferase (HPRT) locus that is almost certainly a result of his exposure to A-bomb radiation. Our key finding is that we detected a very much higher HPRT mutant frequency in the naive (CD45RA(+)) cell component of this individual's CD4 and CD8 T-cell populations than in the memory (CD45RA(-)) cell component of his CD4 and CD8 T-cell populations. This stands in marked contrast to our finding that HPRT mutant frequencies are fairly similar in the na?ve CD45RA(+) and memory CD45RA(-) components of the CD4 and CD8 T-cell populations of three unexposed individuals examined concurrently. In addition we found that the HPRT mutant frequencies were about 30-fold higher in the na?ve (CD45RA(+)) CD4 T cells of the exposed individual than in his memory (CD45RA(-)) cell populations, but that the effect was a little less striking in his CD8 cell populations, where the HPRT mutant frequencies were only about 15-fold higher in his na?ve T-cell pools than in his memory T-cell pools. We further found that 100% of the HPRT mutant cells in both his CD4 and CD8 na?ve cell subsets appeared to have originated from repeated divisions of the initial HPRT mutant stem cell, whereas only 4 of 24 and 5 of 6 mutant cells in his CD4 and CD8 memory cell subsets appeared to have originated from that same stem cell. The most straightforward conclusion may be that the great majority of the T cells produced by this individual since he was 17 years old have remained as na?ve-type T cells, rather than having become memory-type T cells. Thus the T cells that have been produced from the hematopoietic stem cells of this particular A-bomb-exposed individual seldom seem to enter and/or to remain in the memory T-cell pool for long periods. We speculate that this constraint on entry into memory T-cell pools may also apply to unirradiated individuals, but in the absence of genetic markers to assist us in obtaining evidential support, we must await clarifying information from radically different experimental approaches.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary Each ovarian follicle of Triops cancriformis is four-celled; these cells (one oocyte and three nurse cells) are interconnected by cytoplasmic bridges. In the course of differentiation, the nurse cells are early recognizable; they increase in size more than the oocyte and their nuclei contain many nucleoli. For the first time in Arthropoda, yolk globules are reported to be present in nurse cell cytoplasm; these globules arise from the smooth endoplasmic reticulum. The functional significance of the intercellular bridges and the trophic role of the nurse cells are discussed.The authors are grateful to Dr. Bruno Sabelli for his support and to Mr. Francesco Monte for his technical assistance     

Evidence for corticotropin releasing factor (CRF) synthesis in the preoptic nucleus of Xenopus laevis tadpoles

Summary For the study of the hypothalamo-hypophysial system of Xenopus laevis tadpoles, hypothalamic lesions were made by means of the electrocoagulation technique. Lesioning of the ventral region of the preoptic nucleus resulted in a decrease of the number of ACTH cells in the pars distalis of the pituitary gland and in a diminution of the PAS-positive reaction of these cells. In addition, regeneration of the neurosecretory cells of the ventral region of the preoptic nucleus observed 6 weeks after lesioning was accompanied by the reappearance of normal PAS-positive ACTH cells in the pars distalis. It is suggested that the neurosecretory cells of the ventral region of the preoptic nucleus of Xenopus laevis tadpoles are related to the ACTH synthesizing cells, probably by producing CRF.Dedicated to Prof. Dr. med. W. Bargmann on the occasion of his 70th birthdayThe authors thank Prof. Dr. J.C. van de Kamer for his interest, Prof. Dr. P.G.W.J. van Oordt for his many helpful comments, and Messrs. H. van Kooten, E. van der Vlist, J.J. van der Vlis and M.C.A. van Pinxteren for preparing the illustrations     

Somato-dendritic synapses in the paraventricular organ of two anuran species

Summary The ultrastructure of the paraventricular organ ofXenopus laevis andRana esculenta has been investigated. Special attention has been paid to contact regions between nerve cells of the paraventricular organ and other nerve cells.According to the results of the glutaraldehyde-osmium tetroxide fixation as well as the zinc iodide-osmium method, it appears that the perikarya of several nerve cells of the paraventricular organ make synaptic contacts with dendrites of other nerve cells. It is suggested that the presence of the somato-dendritic synapses is a morphological indication for the sensory function of the nerve cells.Thanks are due to Prof. Dr. P. G. W. J. van Oordt for his interest and the many helpful discussions, and to Mr. H. van Kooten and his co-workers for their skilful photographic assistance.     

Giulio Bizzozero: a pioneer of cell biology

The Italian pathologist Giulio Bizzozero began his haematological investigations more than 130 years ago. Among his outstanding achievements was the discovery of the role of platelets in haemostasis and the identification of the bone marrow as the site of production of blood cells. One hundred years after his untimely death, the significance of these, and many more of his findings, is still recognized.     

Immunologic studies with LFA-1- and Mo1-deficient lymphocytes from a patient with recurrent bacterial infections

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

A tribute to Shinya Inoue and innovation in light microscopy

The 2003 International Prize for Biology was awarded to Shinya Inoue for his pioneering work in visualizing dynamic processes within living cells using the light microscope. He and his scientific descendants are now pushing light microscopy even further by developing new techniques such as imaging single molecules, visualizing processes in living animals, and correlating results from light and electron microscopy.     

The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity

Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity. It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA. Here, we confirm that a his::MudJ operon fusion in S. typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures. Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA. Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically. Both environmental repression and novobiocin induction of his require the his attenuator. In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.     

On key lesions and all that: a tribute to Paul Lohman

This paper is a tribute to Paul Lohman at the occasion of his retirement from the position of Professor in the Medical Faculty at the Leiden University in The Netherlands and as Director of its Department of Radiation Genetics and Chemical Mutagenesis. Paul's contributions to the science of genetic toxicology are discussed in the context of more recent insights as to how mammalian cells process DNA damage, and how this may lead to cancer and, possibly, aging. Starting with his work on the characterization of UV-induced DNA repair in cultured cells from xeroderma pigmentosum patients and the development of methodology for monitoring the removal of UV-induced lesions in human cells, the concept of the key lesion is introduced. Among the myriad of DNA lesions that can be induced in DNA as a consequence of exposure to a range of natural or synthetic mutagens, key lesions are the ones responsible for subsequent adverse effects, for example, because they give rise to mutation. The development of methods using immunofluorescence microscopy to detect and identify such key lesions and quantitate them at the single cell level, is one of the highlights of Paul's career. Based on the perceived need to evaluate mutational end points in vivo in relation to specific lesions identified by his immunofluorescence methods, Paul subsequently made crucial contributions to the development of the first transgenic mouse model to measure mutations in chromosomally integrated reporter genes. In parallel to his experimental work, Paul greatly contributed to genetic toxicology at the theoretical level by his work on the development and evaluation of methods for assessment or prediction of risks of exposure to environmental mutagens. Finally, Paul has served the discipline of genetic toxicology in a more administrative role in various ways, both locally as one of the founders of the Medical Genetics Center South-West Netherlands and internationally by playing a prominent role in organizations such as ICPEMC. Here, his numerous contributions to the journal Mutation Research, both as author on many papers and as Executive Managing Editor should not go unmentioned.     

CTLA-4.FasL inhibits allogeneic responses in vivo

CTLA-4.Fas ligand (CTLA-4.FasL), a paradigmatic 'trans signal converter protein (TSCP)', can attach to APC (via CTLA-4 binding to B7) and direct intercellular inhibitory signals to responding T cells (via FasL binding to Fas receptor), converting an activating APC-to-T cell signal into an inhibitory one. Our previous studies established that CTLA-4.FasL inhibits human primary mixed lymphocyte reactions (MLR) and induces alloantigen-specific hyporesponsiveness ex vivo. The present study extends this to an in vivo context. Using splenocytes from MHC-mismatched C57BL/6 and Balb/c mice, we demonstrated that his(6)CTLA-4.FasL, effectively inhibits murine MLR. Moving in vivo, we demonstrated that subcutaneously administered his(6)CTLA-4.FasL modulates the in vivo response of infused allogeneic splenocytes. his(6)CTLA-4.FasL reduces the number of cells in each cell division, and increases the percentage of dead cells in each division. These findings are consistent with an antigen-induced cell death of the alloreactive cells, and bolsters recombinant TCSP promise as a therapeutic for transplantation diseases.     

Immunocytochemical diagnosis of lymphoma from urine sediment

Cytologic studies were done on the urine sediment of a patient with an indolent lymphoma of 20 years' duration. The patient had localized disease in his groins and developed marked swelling in his penis and scrotum and pedal edema shortly after radiotherapy was instituted to the inguinal areas. The urine specimen obtained after chemotherapy showed many large mononucleated malignant cells. These cells were extremely fragile and were best demonstrated by supravital staining of wet preparations. Although morphologic examination could not confidently indicate the origin of these malignant cells, cytochemical and immunocytochemical studies showed them to be monoclonal B lymphocytes. This study suggests that, even under unusually difficult circumstances, a combined cytologic, cytochemical and immunocytochemical approach can be successfully applied for cytodiagnosis.     

Kinematographische Dokumentation einer amitotischen Zellteilung

Summary Identification of argyrophilic cells, in pancreatic islets of normal rabbits, is accomplished by light and electron microscopy in osmium-fixed plastic-embedded tissues.Fixative, pretreatment and pH of silver nitrate solution were essential for the light microscopic study to reveal argyrophilic cells in osmium-fixed plastic-embedded pancreatic islet tissue. The best result was obtained with Dalton's osmium fixation and buffered silver nitrate methanamine solution at pH 9.O. The cytoplasmic granules of argyrophilic cells generally are densely packed but some of the cells show only sparse silver impregnated granules in the cytoplasm. Occasionally there are some non-argyrophilic granular cells in which, after silver impregnation, the cytoplasm appears clear. There are three kinds of cells in the pancreatic islets, i.e., argyrophilic granular cells, non-argyrophilic granular (clear) cells, and beta cells (situated centrally in the islet and stained light yellow in silver impregnated sections).The cells known as argyrophilic cells in light microscopy can be identified as alpha cells in electron micrographs by comparison of consecutive sections of the same cell.The author would like to express his appreciation to professor Roy C. Swan for his generous guidance.     

Combined protocol of cell therapy for chronic spinal cord injury. Report on the electrical and functional recovery of two patients

BACKGROUND: This is a preliminary report on successful results obtained during treatment of two patients with chronic spinal cord injury. The therapeutic approach was based on the generation of controlled inflammatory activity at the injury site that induced a microenvironment for the subsequent administration of autologous, BM-driven transdifferentiated neural stem cells (NSC). METHODS: BM mesenchymal stem cells (MSC) were cocultured with the patient's autoimmune T (AT) cells to be transdifferentiated into NSC. Forty-eight hours prior to NSC implant, patients received an i.v. infusion of 5 x 10(8) to 1 x 10(9) AT cells. NSC were infused via a feeding artery of the lesion site. Safety evaluations were performed everyday, from the day of the first infusion until 96 h after the second infusion. After treatment, patients started a Vojta and Bobath neurorehabilitation program. RESULTS: At present two patients have been treated. Patient 1 was a 19-year-old man who presented paraplegia at the eight thoracic vertebra (T8) with his sensitive level corresponding to his sixth thoracic metamere (T6). He received two AT-NSC treatments and neurorehabilitation for 6 months. At present his motor level corresponds to his first sacral metamere (S1) and his sensitive level to the fourth sacral metamere (S4). Patient 2 was a 21-year-old woman who had a lesion that extended from her third to her fifth cervical vertebrae (C3-C5). Prior to her first therapeutic cycle she had severe quadriplegia and her sensitive level corresponded to her second cervical metamere (C2). After 3 months of treatment her motor and sensitive levels reached her first and second thoracic metameres (T1-T2). No adverse events were detected in either patient. DISCUSSION: The preliminary results lead us to think that this minimally invasive approach, which has minor adverse events, is effective for the repair of chronic spinal cord lesions.     

A long-awaited discovery: hypoxia prevents mouse cells from undergoing spontaneous p53-dependent transformation

A recent publication by Phinney and his associates (1) has presented a discovery for which the field of cell biology has been waiting for more than half a century: a protocol that makes it possible to expand mouse cells in culture without the cells spontaneously transforming. Amazing though it seems, the secret is simply to grow the cells under hypoxic conditions.     

Intracranial and intraspinal dissemination of an ACTH-secreting pituitary tumor.

A case of a 29-year-old man with an ACTH-producing pituitary tumor disseminated into the subarachnoid space is described. After total adrenalectomy for Cushing's disease at the age of 15, Nelson's syndrome developed. Transsphenoidal adenomectomy at 17 and 21 years of age, pituitary irradiation and medical therapies with sodium valproate, baclofen and bromocriptine failed to lower his plasma ACTH level. Multiple intracranial and intraspinal tumors associated with the symptoms of left hemiparesis developed. The removal of a tumor grown at the level of C1-3 was performed with successful palliation of his symptoms. Histologically, the tumor cells showed sinusoidal, papillary and diffuse patterns with a preponderance of the former over the latter two, although the papillary pattern predominated in the primary pituitary tumor. Immunohistochemical analysis demonstrated most cells to be positive for ACTH in the metastatic tumor as well as the primary adenoma. The clinical significance of his course is discussed with a review of 11 reported cases with metastatic ACTH-producing pituitary tumors.     

Unusual response to bifunctional alkylating agents in a case of Fanconi anaemia

Summary Chromosomal breakage frequencies were determined in Fanconi anaemia (FA) blood cultures treated with various concentrations of the polyfunctional alkylating agents mitomycin C, diepoxybutane, and cis-platinum(II)-diammine-dichloride, for which FA cells have a characteristic hypersensitivity. At concentrations that hardly affected control cultures, three out of four patients tested exhibited a concentration-dependent increase of cells with aberrant chromosomes, with a concomitant increase in the number of chromosomal aberrations per aberrant cell. The fourth patient, a 22-year-old male, was exceptional because with all three clastogens only 40% of his cultured cells exhibited a typical concentration-dependent response, while 60% of his cells responded like those from normal healthy controls. The possible nature and significance of this unusual response is discussed.