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Biomedical subjects

M Shalit

Publications and source records attributed to M Shalit.

At least 37 records · Page 2Linked to original sources
PubMed1995

Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes.

Chronic granulomatous disease (CGD) is a rare recessive disorder caused by defects in the NADPH oxidase enzyme complex of phagocytes (neutrophils, eosinophils and monocytes). CGD phagocytes fail to produce superoxide and other reactive oxygen species following cell activation (Malech, 1993). The products of oxidase activation can be measured in individual cells by flow cytometry using specific fluorescent probes that increase fluorescence upon oxidation (Trinkle et al., 1987). This approach can be used to confirm a diagnosis of CGD, and to detect the normal/abnormal phagocyte mixture that characterizes the X-linked CGD carrier state. Three fluorescent probes have been described as useful for this purpose: 2'7'-dichlorofluorescin diacetate (DCF) (Bass et al., 1983), 5,6-carboxy-2'7'-dichlorofluorescein diacetate, bis(acetoxymethyl) ester (C-DCF) (Hockenbery et al., 1993) and dihydrorhodamine 123 (DHR) (Rothe et al., 1988; Kinsey et al., 1987). A direct comparison between these three probes has not been reported. In this study we performed a direct comparison between these three probes, evaluating their ability in flow cytometric analysis to maximize fluorescent separation between activated CGD patient and normal granulocytes. Using a whole blood technique with phorbol myristate acetate (PMA) as an activator, it was found that DHR loaded normal granulocytes had a fluorescence intensity which, upon activation, was 48-fold higher than that of C-DCF loaded granulocytes and seven-fold higher than DCF loaded granulocytes (P < 0.001). Use of sodium azide to decrease the catabolism of H2O2 enhanced the fluorescence of DCF by 140%, C-DCF by 45% and DHR by 25%, suggesting that DCF is primarily sensitive to H2O2. DCF and DHR were then evaluated for sensitivity in the detection of small percentages of normal cells in a CGD/normal granulocyte mixture. Normal sub-populations as small as 0.1% could clearly be distinguished using DHR, while DCF was insensitive at this level. Based on these findings, we used DHR in an effort to detect normal granulocytes in a CGD patient following therapeutic granulocyte transfusion. We were able to detect normal granulocytes in the circulation for up to 18 h after transfusion. With these data we show that DHR is the most sensitive flow cytometric indicator for the detection of oxygen reactive species in activated granulocytes and is the best probe for evaluating CGD patients and carriers. In addition, our data suggest that DHR is a useful tool for monitoring circulating normal granulocytes in CGD patients following transfusion, and potentially will be a sensitive probe for assessing the success of such future technologies as gene therapy for CGD.

Flow Cytometry↗
PubMed1995

Modulation of growth and differentiation of eosinophils from human peripheral blood CD34+ cells by IL5 and other growth factors.

Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34+ cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G-CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3+GM-CSF+IL5), the addition of interferon-alpha or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-gamma, tumor necrosis factor-alpha, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10(4) cells plated at Day 3 and 134 colonies/10(4) cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5 independent. Using reverse-transcriptase polymerase chain reaction, the mRNA encoding the eosinophil-specific protein eosinophil peroxidase (EPO) was not detected in Day 0 CD34+ cells, but was demonstrated by Day 3 of culture. We conclude that within 3 days of culture, peripheral blood CD34+ cells can become committed to the eosinophil lineage as demonstrated by responsiveness to IL5 and production of EPO transcripts.

Antigens, CD↗
PubMed1995

Use of epinephrine for anaphylaxis by emergency medical technicians in a wilderness setting.

STUDY OBJECTIVE: To describe a case series of emergency medical technician-basic (EMT-B)-administered epinephrine for anaphylaxis in a wilderness setting. DESIGN: Case series of patients in anaphylaxis who received epinephrine subcutaneously from EMT-Bs. SETTING: National park rural/wilderness emergency medical service system covering 863,000 acres and serving approximately 2 million annual visitors. PARTICIPANTS: Prehospital care providers were National Park Service rangers with EMT-B training. Patients in the series were visitors to Sequoia and Kings Canyon National Parks with anaphylaxis resulting from insect stings. INTERVENTION: Subcutaneous administration of epinephrine by EMT-Bs trained in recognition, understanding, and treatment of anaphylaxis. RESULTS: Eight patients with anaphylaxis resulting from Hymenoptera stings, from June 1992 through September 1993, received EMT-B-administered epinephrine. All patients improved clinically after treatment with epinephrine within 25 minutes. No major side effects occurred. CONCLUSION: Our data suggest that EMT-B-administered epinephrine is safe when used by EMT-Bs in the rural/wilderness setting, with appropriate physician supervision. Further study in large trials will be required to demonstrate safety and efficacy.

Adolescent↗
PubMed1995

Challenge of mast cells with increasing amounts of antigen induces desensitization.

BACKGROUND: In vivo rush desensitization is a procedure widely employed to quickly desensitize allergic patients by administering increasing doses of the offending antigen at short intervals. The mechanism(s) underlying this process and the possible role of mast cells in it have not been well delineated. OBJECTIVE: To define an ex vivo model for rush desensitization utilizing the mast cell-fibroblast coculture system. METHODS: Rat peritoneal mast cells were cultured on 3T3 fibroblasts and were preincubated with saturating or suboptimal concentrations of IgE anti-DNP antibodies. Mast cells were then repeatedly challenged at 30 min intervals, with increasing amounts (0.001-100 ng/mL) of the antigen DNP-HSA, in the presence of calcium ions. Parallel control cultures were stimulated only once by each antigen concentration. In another set of experiments, mast cells were repeatedly activated with increasing concentrations (0.1-1000 ng/mL) of compound 48/80. Supernatants and cell sonicates were assessed for histamine content and the percentage of histamine released was calculated. RESULTS: When saturating concentrations of IgE anti-DNP antibodies were used, mast cells challenged repeatedly with DNP-HSA did not release significant amounts of histamine up to an antigen concentration of 1 ng/mL. At this stage they were partially desensitized, releasing only 108.3 +/- 17.1 ng histamine/plate (7.9 +/- 0.8%). A marked desensitization was observed at optimal antigen concentration (100 ng/mL), where experimental mast cells released only 45.6 +/- 10.9 ng/plate, compared with 661.9 +/- 48.2 ng/plate in firstly challenged cultures. Desensitization was probably not due to mast cells histamine depletion, since the cells still contained large amounts of histamine (579.5 +/- 108.6 ng/plate) at the end of the procedure. A similar pattern of desensitization was observed when mast cells were preincubated with a suboptimal concentration of IgE antibodies. Activation of mast cells with increasing amounts of the IgE-independent secretagogue, compound 48/80, did not cause desensitization since at each concentration both repetitively challenged and control cultures released similar amounts of histamine. Furthermore, challenge of antigen-desensitized mast cells with compound 48/80 caused the release of 75.9 +/- 4.9% histamine comparable to the percentage of histamine released from controls (79.5 +/- 6.7). CONCLUSION: Repeated exposure of mast cells to gradually increasing amounts of antigen induces their refractoriness. This observation would suggest a role for mast cells in rush desensitization procedure in vivo. Our coculture system may serve as a useful model for studying this process.

3T3 Cells↗
PubMed1995

Imbalance of immune responses in patients with chronic and widespread fungal skin infection.

Fungal infections of the skin are caused by dermatophyte fungi. Infections can be acute and inflammatory or chronic and non-inflammatory; it is believed that cell-mediated immunity is the cornerstone of host defence and is instrumental in the eradication of the infection. We describe here parameters of the immune response of a group of patients who although not immunocompromised, suffered from widespread and chronic infections. All patients lacked a specific delayed-type hypersensitivity (DTH) response; however, their in vitro lymphocyte proliferation in response to Trichophyton rubrum extract and to other fungal antigens was normal. The patients were not atopic by clinical history, and yet had high levels of non-specific IgE and of T. rubrum-specific IgG4. Taken together, the results of this study suggest that the group of patients studied suffered from an immune imbalance which has characteristics of a Th2-type response.

Adult↗
PubMed1994

Giant cranial base tumours.

Thirty-three patients with giant (diameter > or = 4.5 cm) cranial base tumours who underwent surgery at the Hadassah Hospital over the last ten years are described. Twenty-three of the patients had meningiomas, 4 neurinomas, one giant cell tumour, one haemangiopericytoma, and 4 had malignant meningiomas. Four tumours were at the cerebellopontine angle, 9 within the anterior cranial fossa, 8 petroclival, 8 on middle fossa floor, and 4 along the sphenoid ridge. The average pre-operative symptom duration was 31 months, range 3-180 months. Nineteen patients had a radical tumour resection, 10 subtotal, and 4 a partial resection on an average 1.7 operations per patient. The mean follow-up period from the first operation was 39 months (range 2-120). There was no mortality peri-operatively or during the follow-up period. The mean pre-operative Karnofsky score was 68 and at the last follow-up 76. There was no correlation between histology and degree of resection, complications, or status at last follow-up. The best resections (92% radical) and outcome (mean Karnofsky 92) with the least number of operations (mean 1.4) were in the anterior fossa and along the sphenoid wing. The patients requiring the most operations (mean 2.1), having the smallest percentage of radical resections (25%) and the least favourable outcomes (mean Karnofsky 52) were those with petroclival tumours. Patients with giant cranial base tumours have a good overall long-term prognosis, but especially those with petroclival tumours challenge us to improve our techniques.

Adolescent↗
PubMed1994

Mast cells in experimental allergic encephalomyelitis: characterization, distribution in the CNS and in vitro activation by myelin basic protein and neuropeptides.

Mast cells (MC) have been implicated in the pathogenesis of experimental allergic encephalomyelitis (EAE). In order to further evaluate their role, several morphological and functional studies were performed. Semiquantitative counts of histological sections showed a significant reduction in MC numbers in EAE brains. In addition, a higher proportion of EAE MC (about 50-70%) appeared degranulated compared with about 20% degranulation in controls. Central nervous system (CNS) MC exhibited staining properties of connective tissue MC and about 98% of them, both in diseased and control rats, were located in the thalamus. They were not present in the spinal cord and did not relate to EAE lesions. In vitro incubation of peritoneal MC (of connective tissue phenotype) with either MBP, or with neuropeptides such as substance P or bradykinin resulted in release of beta-hexosaminidase and histamine. The latter responses were similar in both EAE and control rats. It is suggested that the decrease in number and in granular content of CNS MC in EAE may reflect prior in vivo activation. The fact that MC were activated by MBP and by neuropeptides in vitro suggests a possible mechanism of MC activation in EAE.

Animals↗
PubMed1993

[Delayed pressure urticaria: therapeutic response to cetirizine].

We describe a 39-year-old man who suffered from delayed pressure urticaria for 2 years. His severe symptoms did not respond to antihistamine (H1 and H2) treatment and corticosteroid therapy was given despite severe side-effects. A new, nonsedating antihistamine drug, cetirizine, relieved most of his symptoms, enabled him to be weaned from corticosteroid therapy, and restored his former quality of life.

Adult↗
PubMed1993

Proliferation and functional responses of bone marrow-derived mast cells after activation.

To investigate the behavior of mast cells following activation we have studied IL-3-dependent mouse bone marrow-derived mast cells (BMMC). Proliferation of BMMC presensitized with IgE anti-DNP antibodies was assessed after immunological stimulation with DNP-HSA. Proliferative potential of activated BMMC, as detected by [3H]thymidine incorporation, was similar to that of control BMMC during the first 6 days after activation while on Day 9 activated cells incorporated more [3H]thymidine. Both activated and control BMMC numbers slightly decreased 3-4 days after challenge with antigen and by Day 5 started to increase, reaching a maximum at Day 9. Immunologic activation of BMMC presensitized with IgE antibodies led to a profound loss of responsiveness to rechallenge with the same antigen 2-24 hr and 3-5 days later (70-100% inhibition of beta-hexosaminidase release). After 12 days cells were still partially unresponsive (20% inhibition). On the other hand, when BMMC were incubated again with IgE prior to rechallenge, desensitization was less pronounced and was resolved by Day 12. Activation of BMMC with an optimal concentration of thrombin induced only a partial unresponsiveness upon rechallenge 2-4 hr after activation (30% inhibition of beta-hexosaminidase release), while activation with calcium ionophore did not induce any desensitization. In both control and experimental BMMC cell-associated histamine remained stable for 12 days after challenge. We conclude that BMMC undergo a period of unresponsiveness which is more pronounced after immunological activation in contrast to nonimmunological ones, similarly to what previously shown for rat peritoneal mast cells.

Animals↗
PubMed1993

Interaction between mast cells and glial cells: an in vitro study.

Brain mast cells (MC) are located in close proximity to glial cells and it has been suggested that they belong to the connective tissue phenotype. To determine whether the local microenvironment provided by glial cells can influence mouse bone marrow-derived MC (BMMC), the putative counterpart of mucosal MC, we co-cultured these two cell types. BMMC numbers, morphology, histochemical properties and histamine content as well as glial cell morphology and function were evaluated up to 21 days. Our data indicate that BMMC adhere, proliferate, survive and can be activated to release histamine on the glial cell monolayers without changing their phenotype. Co-cultured glial cells preserve their morphological appearance and function throughout the culture period. These data indicate that central nervous system (CNS) glial cells do not induce phenotypic changes in BMMC and do not interfere with their viability and function.

Animals↗
PubMed1993

Heteroduplex strand-specificity in restriction-stimulated recombination by the RecE pathway of Escherichia coli.

The RecE recombination pathway is active in Escherichia coli recB recC sbcA mutants. To isolate and characterize products and intermediates of RecE-mediated, break-induced, intramolecular recombination, we infected recB recC sbcA mutants, expressing EcoRI endonuclease, with chimeric lambda phages that allow EcoRI-mediated release of cloned linear recombination substrates. Substrates with direct terminal repeats recombined to yield a circular product with one copy of the repeated sequence. Some recombinants were heteroallelic for the recombining markers. Markers distant to the break were recovered in the circular product at a higher frequency than markers close to the break. To examine the heteroduplex structures that may have yielded the heteroallelic recombinants, nonreplicative substrates were employed. Some of the nonreplicative recombination products contained heteroduplexes, with a strong bias for paired strands ending 3' at the break. This strand bias in heteroduplex formation is consistent with recombination models that postulate homologous pairing of protruding 3' single-stranded ends.

Base Sequence↗
PubMed1993

Diminished actin polymerization by neutrophils from newborn infants.

During the newborn period, abnormalities of neutrophil (PMN) function predispose infants to serious bacterial disease. Actin is a major contributor to PMN shape change and motile behavior. To determine the mechanism underlying defects in newborn granulocyte polarity and chemotaxis, we investigated actin polymerization by cord blood PMN from healthy term infants and adult controls. F-actin (filamentous) content was quantified in the resting state and after stimulation by fluorescence-activated cell-sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells. PMN from newborn infants demonstrated similar basal F-actin levels when compared with adults. N-formyl methionyl leucyl phenylalanine induced a marked increase in actin polymerization that was maximal at 30 s in both neonates and adults and that then declined slowly (depolymerization) over the following 10 min. However, the F-actin content of PMN from newborn infants was significantly diminished when compared with adults at 30 and 60 s after N-formyl methionyl leucyl phenylalanine stimulation (p < 0.05). Both the rate and dose response of N-formyl methionyl leucyl phenylalanine-induced actin polymerization were similar for adult and neonatal PMN. PMN from newborn infants also demonstrated significantly diminished actin polymerization when compared with adults 60 s after stimulation with platelet-activating factor (p < 0.05). Decreased concentrations of F-actin may help explain the observed abnormalities of PMN polarity and chemotaxis in healthy newborn infants.

Actins↗
PubMed1993

Mast cells retain their responsiveness upon continuous and repetitive exposure to antigen.

To assess the effects of prolonged and repetitive exposure of antigen on mast cells (MC) we utilized our co-culture system in which rat peritoneal MC are kept viable and functionally active on 3T3 fibroblasts. MC were presensitized with IgE anti-dinitrophenyl-human serum albumin (anti-DNP-HSA), activated with the relevant antigen (DNP-HSA) (69.4% histamine release) and incubated for 7 days in medium alone or medium containing the antigen with or without Ca2+ ions. During the prolonged incubation MC released only small amounts of histamine (20-26 ng/plate/day) under all the different culture conditions. Moreover, these MC were not activated after each medium replacement with fresh antigen. When activated MC cultured in medium containing antigen and Ca2+ were presensitized prior to a second challenge performed on days 1, 2, 5 and 7, they released 35-45% histamine but were still partially desensitized compared to initially challenged MC. Immunofluorescence studies demonstrated that the MC unresponsiveness to a second challenge could be partially attributed to lack of membrane-associated IgE antibodies. Repetitive activation (four times) of MC at daily intervals did not abrogate their releasing capacity although the intracellular histamine content was diminished. When repetitive activation was performed 2 and 4 hr after the first challenge, a more intense MC refractoriness was observed (release of 17.3% and 21.1% histamine respectively versus 67.7%). It is suggested that MC activation occurs even after continuous exposure to antigen and that MC can respond to repetitive immunological stimulation.

Animals↗
PubMed1993

Bilateral pulmonary artery aneurysms in Behçet's disease.

A patient presented with fever, haemoptysis and large bilateral perihilar masses. Histology proved that these were pulmonary artery aneurysms due to pulmonary vasculitis, as a manifestation of Behçet's disease. It is suggested that a routine dynamic computed tomographic (CT) scan should be performed in the evaluation of such cases.

Adolescent↗
PubMed1993

Episodic angioedema with eosinophilia: a case associated with T cell activation and cytokine production.

Episodic angioedema with eosinophilia is a rare syndrome, characterized by periodic attacks of fever, myalgia, angioedema, and oliguria, associated with an increased eosinophil count. The pathogenesis of this disorder is incompletely understood. We describe an additional patient with episodic angioedema and eosinophilia, and review the literature regarding the clinical features and treatment of this disease. Previous hypotheses regarding disease mechanisms are discussed. In our patient, we found high basal levels of interleukin-1 (IL-1) and soluble interleukin-2 receptor (sIL-2R), with further increases in levels of both mediators during attacks. We suggest that IL-1 and T cell activation may play a role in the pathophysiology of episodic angioedema with eosinophilia.

Adult↗