Vigabatrin and behaviour disturbances.
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Aggression↗
Biomedical subjects
M K Robinson
Publications and source records attributed to M K Robinson.
Use of an optimized in vitro lymphocyte blastogenesis assay to detect contact sensitivity to nickel sulfate in mice.
This study was designed to determine whether an optimized in vitro lymphocyte blastogenesis assay for identification of strong contact sensitizers would also detect sensitization to the weaker, clinically relevant allergen nickel sulfate (NiSO4). Though NiSO4 is effective in eliciting allergic skin reactions in patients sensitized to nickel-containing metals, it has been difficult to assess its potential to induce sensitization using standard or developmental in vivo animal tests. In vitro lymphocyte blastogenesis has been useful in the diagnosis of nickel allergy in humans, but has not been applied to predictive testing in animals. We used a previously optimized lymphocyte blastogenesis assay to determine whether lymphocytes from NiSO4-treated mice would exhibit NiSO4-specific proliferation and whether this would correlate with an in vivo ear swelling response. BALB/c mice given repeated open induction applications of NiSO4 were ear challenged, then lymphocytes from the draining nodes were cultured with Langerhans cell-enriched epidermal cells (EC), EC + soluble NiSO4, or NiSO4-modified EC (modified by preincubation with NiSO4). The NiSO4-modified EC stimulated significant NiSO4-specific proliferation. EC + soluble NiSO4 stimulated a nonspecific blastogenic response in lymphocytes from both NiSO4-treated and naive mice. There was no ear swelling response to NiSO4 using standard challenge procedures. However, exaggeration of the challenge procedure by gently abrading the ears just prior to NiSO4 application resulted in significant ear swelling, thereby supporting the conclusion that the in vitro results were indicative of in vivo sensitization.
Animals↗
A review of the Buehler guinea pig skin sensitization test and its use in a risk assessment process for human skin sensitization.
The Buehler test is a valuable procedure for screening the sensitization potential of chemicals prior to human exposure. Our experience of over 20 years has shown it to be effective in detecting strong, moderate, and most weak sensitizers. The topical exposure inherent in the Buehler test allows it to be utilized to investigate dose responses, cross reactivity between structurally related chemicals, and the sensitization potential of contaminants in raw material mixtures. For safety assessment purposes, Buehler test results provide an initial indication of the sensitization potential of the material in question under relevant, but exaggerated, exposure conditions. These results can be compared to results on benchmark chemicals to assess sensitization risk for subsequent human exposure. Optimizing the sensitivity of the Buehler test requires adherence to the published methodology and proper interpretation of the challenge and rechallenge data obtained. Adjuvant-type test methods are generally considered to be more sensitive than topical methods. However, when done properly, topical test procedures such as the Buehler test or the open epicutaneous test can accurately detect most chemicals with any realistic potential for sensitizing humans by the topical route. Moreover, from a risk assessment perspective, these topical tests avoid the problems of overestimating the weak sensitization potential of many topically applied materials or underestimating the sensitization potential of very strong sensitizers; both are potential concerns with invasive adjuvant-type test methods. The Buehler test or other topical test methods are particularly valuable for comparative sensitization risk assessment since human sensitization data on benchmark materials are all derived from topical exposure. The risk assessment is developed by comparing the guinea pig data on the new material versus relevant benchmark chemicals or formulations and also by evaluating the existing human sensitization data on the benchmark material. These data are then utilized to predict human sensitization risk from topical exposure to the new material. Confirmation of human safety can be derived from human repeat insult patch testing (HRIPT) and other clinical tests such as the product use test and the diagnostic patch test. Utilized in this manner, the Buehler test is an integral component of an overall skin sensitization safety assessment program for a new chemical or product formulation.
Animals↗
Immunosuppressive effects of clonidine on the induction of contact sensitization in the balb/c mouse.
The clonidine transdermal therapeutic system (clonidine-TTS) has been associated with a significant incidence of allergic contact sensitization. This incidence was not predicted by premarket skin sensitization testing in animals or humans. One possible explanation lies in recent findings in guinea pigs that clonidine exposure could inhibit the elicitation of skin reactions to unrelated strong contact sensitizers. However, these studies also showed that clonidine pretreatment did not appear to affect the induction of contact sensitization. On this basis, we sought to specifically evaluate the induction phase of sensitization to clonidine as an alternative means of assessing its sensitization properties. The method selected was the assay of in situ lymphocyte proliferation in lymph nodes draining the sites of clonidine exposure, a method recently promoted as an alternative means to assess contact allergenic potential. Utilizing various induction application techniques and regimens, we were consistently unable to demonstrate clonidine's allergenic potential through such an assessment of lymphocyte proliferation. We were also unable to demonstrate sensitization by in vivo ear swelling or in vitro lymphocyte blastogenesis assay techniques. However, a subsequent assessment of the effect of clonidine exposure on the induction of sensitization to unrelated strong contact allergens demonstrated a consistent 40-70% inhibition of the proliferative response to the contact allergens oxazolone and trinitrochlorobenzene. This was similar to the degree of suppression produced by the corticosteroids fluocinonide and hydrocortisone when they were tested at 80 and 10 times lower concentrations. In addition, we observed a comparable inhibition of the ear swelling response to oxazolone. These data extend our knowledge of the immunomodulatory effects of clonidine and offer additional mechanistic insights into the failure of short-term predictive patch-test methods to detect this chemical's potential to induce allergic contact sensitization.
Animals↗
Value of the cutaneous basophil hypersensitivity (CBH) response for distinguishing weak contact sensitization from irritation reactions in the guinea pig.
Numerous studies of the histology of allergic contact dermatitis reactions to potent allergens in guinea pigs and humans have indicated that there is significant tissue infiltration with basophilic leukocytes. In this study we determined whether this histologic finding could be of value in distinguishing weak sensitization reactions from primary irritation, thereby aiding in the predictive identification of weak or moderate contact allergens. Guinea pigs were sensitized by the Buehler test method. Skin reactions were graded 24, 48, and 72 h post-challenge with duplicate patch sites biopsied at the 24- or 72-h grading timepoints. The biopsies were fixed, embedded in glycol methacrylate, thin sectioned, and Giemsa stained. The number of basophils per 400 leukocytes were counted along the upper dermis just below the dermal/epidermal junction. Challenge patch sites from animals sensitized to a relatively low dose of the strong contact allergen, oxazolone, were compared with patch sites from animals challenged only with a strong irritant, sodium lauryl sulfate (SLS). Compared to normal skin (7.5 +/- 1.0 basophils/400 leukocytes +/- SEM) only the oxazolone patch sites showed significant basophil infiltration (36.8 +/- 6.5), despite the fact that the skin reactions to the low oxazolone challenge dose were relatively weak. SLS patch sites showed no basophil infiltration above normal skin levels (4.8 +/- 0.9). Subsequent blinded studies compared weak/moderate presumptive sensitization reactions (as defined by accepted visual skin grading criteria) to various chemicals (citronellal, vanillin, cinnamic aldehyde, and ethylenediamine) to primary irritation reactions to the same chemicals. In each case, low-challenge-dose sensitization sites on previously treated (induced) animals showed mean basophil infiltration (range, 11.9-69.2 basophils/400 leukocytes) significantly greater than higher-dose irritant reactions (range, 1.6-13.3). The range for normal skin was 0.2-10.2 and the range for strong patch reactions to higher concentrations of oxazolone was 59.8-209.3. These data strongly indicate that light-microscopic quantitation of the CBH response can be used to distinguish relatively weak to moderate contact sensitization reactions from primary irritation reactions to the same chemicals.
Animals↗
Inorganic mercury transport in the proximal tubule of the rabbit.
Inorganic mercury transport was studied in the S1, S2, and S3 segments of the isolated perfused proximal tubule of the rabbit. The concentration of mercury in the perfusate was 18.4 microM. At this concentration all three segments of the proximal tubule underwent degenerative changes that proceeded to cellular necrosis at the end of the tubule which was attached to the perfusion pipet. This pathological process progressed along the tubule for approximately 200 microns. The remainder of the tubule, to the collection pipette, remained intact and free of any pathological changes. In examining the transport of mercury under these condition, it was found that, on average, the S1, S2, and S3 segments all removed inorganic mercury from the luminal fluid at approximately 140 fmol min-1 mm-1. The transport of mercury, as measured by the appearance of 203Hg in the bathing solution, was 80% lower than the removal of 203Hg from the luminal fluid. The mercury appearing in the bath could be accounted for by passive leakage through the necrotic portion of the tubule in the S1 and S2 segments, but not in the S3 segment. Leakage could account for only 16.2% of the transepithelial movement of inorganic mercury in the S3 segment. Inorganic mercury taken up by the tubule (92%) was primarily associated with the structural proteins of the tubular epithelial cells, while very little (8%) was found in the tubular extract. The toxicity of inorganic mercury was determined by titration. Perfusion with 1 microM inorganic mercury produced necrosis. The pathological features appeared to be the same as those resulting with 18.5 microM inorganic mercury.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Identification of haptoglobin alpha-2FF variants in mid-trimester maternal serum as potential markers for Down syndrome.
Exploratory protein analysis in mid-trimester maternal sera by 2-dimensional electrophoresis and image analysis was performed to determine the differences between mothers carrying fetuses with Down syndrome (DS) and control mothers. Nine haptoglobin alpha-1 and alpha-2 (Hp1 and Hp2) variants were detected. Three apparent isoforms of the Hp2FF protein having the same relative charge but different Mr's were detected, with the two smaller variants--Hp2FF (-0.7k) and Hp2FF (-1.3k)--believed to be first reported here. Ninety-two percent of the cases (n = 83) had the Hp2FF (-0.7k) protein, and 61% had Hp2FF (-1.3k). The geometric mean concentration of Hp2FF (Mr = 18.0 kd) was found to be significantly increased (P less than 0.01) in mid-trimester sera from cases (n = 27) compared to controls (n = 56). When the concentrations of the three Hp2FF isoforms were added, the cases had larger sums on average than controls (P less than 0.01).
Age Factors↗
A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli.
A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.
Alkaline Phosphatase↗
A risk assessment process for allergic contact sensitization.
This review describes an approach that has been used to assess the skin sensitization risk of new chemicals and product formulations prior to launching the new chemical or product on the market. The risk assessment process utilizes a comparative toxicological approach, in which data on the inherent toxicity of a material, and the exposure to it through manufacturing or consumer use or foreseeable misuse, are integrated and compared with data generated by 'benchmark' materials of similar chemistry or product application, or both. This approach has been valuable in providing an accurate assessment of the skin sensitization potential for a wide range of consumer products and pharmaceuticals, ranging from products with a very transient skin exposure (e.g. some paper products), to cosmetics, to products whose ingredients may be deposited on fabrics and thus result in chronic skin exposure. The risk assessment process described includes both guinea-pig (Buehler) and human skin sensitization test methodologies to evaluate inherent toxicity under relevant epicutaneous exposure conditions. Alternative guinea-pig test methods have been reported to be more sensitive than the Buehler method, particularly those employing intradermal injection of the test material in Freund's complete adjuvant (e.g. maximization test). However, by bypassing the skin barrier at induction, these methods can overstate the sensitization risk of epicutaneous exposure to weak sensitizers (Andersen and Hamann, 1983 and 1984; Matsushita et al., 1975a,b), and can understate the risk to very strong sensitizers possibly through tolerance induction (Buehler, 1985). In addition, materials are tested and classified at concentrations that may not be relevant to anticipated human exposure. The Buehler guinea-pig test data are important in assessing skin sensitization risk in the early phases of product development, where human exposure can be limited, controlled and monitored (e.g. manufacturing employees). The Buehler test can often define consumer skin sensitization risk; however, the ultimate consumer skin safety assessment should in general be developed through a series of controlled human tests; the human repeat insult patch test and, when necessary, the provocative or extended product use tests. Post-market monitoring of skin-related consumer comments is the final phase of the data gathering process. These results can be used to assess further each product and to provide valuable feedback to confirm the validity of the overall risk assessment process. Risk assessment for skin sensitization potential is seldom a simple process.(ABSTRACT TRUNCATED AT 400 WORDS)
Consumer Product Safety↗
Optimization of an in vitro lymphocyte blastogenesis assay for predictive assessment of immunologic responsiveness to contact sensitizers.
Current methods for the predictive and diagnostic assessment of contact sensitization rely on the visual scoring of skin reactions. Predictive animal tests, generally using guinea pigs, require a relatively large number of animals to produce a sufficient database for interpreting skin reaction scores. In vitro assays have the potential of being more quantitative than skin testing and, if so, would require fewer animals. However, although in vitro assays are commonly used to study the cellular immune response to strong contact sensitizers, there has been little effort to validate them for predictive assessment purposes. We have optimized an in vitro lymphocyte blastogenesis assay for detecting the response of mouse lymphocytes to strong contact sensitizers with the eventual objective of applying this assay to moderate and weak sensitizers as well. Lymph node lymphocytes from mice sensitized to the strong contact allergens, dinitrochlorobenzene (DNCB), dinitrofluorobenzene (DNFB), or trinitrochlorobenzene (TNCB), responded [greater than or equal to 12,000 counts per minute (CPM) above background] when cultured with water soluble chemical analogues, di- or trinitrobenzene sulfonic acid (DNBS or TNBS). However, the strong sensitizer, oxazolone (OXAZ), has no water soluble analogue and lymphocytes from mice sensitized to OXAZ responded poorly in vitro (less than 2000 CPM) to an ethanol-solubilized OXAZ preparation in spite of very strong in vivo sensitization (ear swelling assay). To increase the assay sensitivity, for OXAZ, we modified the antigen presentation conditions by using 1) solubilized antigen-modified adherent spleen cells, 2) dendritic cells from the draining lymph nodes of antigen painted mice, and 3) antigen-modified Langerhans cell-enriched cultured epidermal cells (EC). These approaches increased OXAZ-directed responses to greater than 7000, greater than 20,000, and greater than 100,000 CPM, respectively, under culture conditions optimized for cell density, responder: stimulator cell ratio, culture duration, and responder cell type. Our results represent a first attempt to directly modify cultured epidermal cells with OXAZ and use these cells to stimulate OXAZ-directed blastogenesis in microtiter plate cultures. This optimized assay is now under evaluation for predictive assessment of contact sensitizers relevant to occupational and consumer exposures.
Animals↗
Translational coupling in the threonine operon of Escherichia coli K-12.
In an attempt to express the two distal genes of the Escherichia coli threonine operon, the majority of the first gene in the operon, thrA, was removed and a series of transcriptional fusions were constructed placing the thrB and thrC genes downstream of either the trp or hybrid tac promoter. Analysis of the proteins produced by cells containing these fusions revealed that although the distal gene, thrC, was efficiently expressed, the proximal gene, thrB, was not expressed at a detectable level. A translational fusion was constructed which fused the cat gene in phase to the last 800 base pairs of thrA followed by thrB and thrC. Cells containing this fusion produced high levels of both the thrB and thrC gene products, showing that translation of thrB requires translation through thrA; thus, thrA and thrB are translationally coupled. In addition, it was found that a sequence between 220 and 57 base pairs before the start of thrB was necessary to allow translational coupling to occur.
Carbon-Oxygen Lyases↗
Peripheral vascular disease. Rheologic variables during controlled ischemia.
The quantitative and qualitative behavior of hemorheologic factors both at rest and after treadmill exercise in 30 male patients with stage II peripheral vascular disease compared with 20 sex- and age-matched healthy controls have been studied. The aim of our study was to identify functional rheologic markers for peripheral vascular disease. At rest, whole blood viscosity (corrected for hematocrit at both high and low shear rates), fibrinogen levels (4.23 +/- 1.39 vs. 3.23 +/- 1.5), and white blood cell count (7.05 +/- 1.25 vs. 6.03 +/- 1.28) were significantly different between patients and controls. After treadmill exercise, white blood cell counts increased in both patients and controls, whereas only the filterability of mononuclear leukocytes showed a significant variation in the patient group (5.47 +/- 1.54 vs. 7.26 +/- 2.00, p less than 0.002). In this group, mononuclear filterability improved during the recovery period. The results suggest a relation between exercise-induced ischemia of the lower limb and mononuclear filterability in patients with peripheral vascular disease. Mononuclear filterability could be a functional rheologic marker for peripheral vascular disease.
Blood Component Removal↗
Effects of polybrominated biphenyls upon rat urinary protein patterns as detected by two-dimensional electrophoresis.
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Animals↗
The fundamental and diagnostic role of T and Tn antigens in breast carcinoma at the earliest histologic stage and throughout.
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Adenocarcinoma↗
Investigation of the instability of plasmids directing the expression of Met-prochymosin in Escherichia coli.
The causes of the instability of a multicopy plasmid, pCT70, which directs the expression of calf prochymosin in Escherichia coli, were investigated. Plasmid pAT153 and its derivative, pCT54, were stable for more than 90 generations in continuous culture with glucose limitation. The multicopy plasmid pCT66, which expressed very low levels of prochymosin due to poor translational efficiency, and low copy number plasmids which efficiently expressed the prochymosin gene, were also stable. These results indicated that high level translation of the recombinant gene was the cause of the instability of pCT70. The maximum specific growth rate of E. coli(pCT70) was reduced by 30% compared with E. coli(pCT66). To fulfil the requirements of a production system, a dual origin plasmid with controllable copy number was developed. Both this plasmid (pMG165) and a derivative which contained the prochymosin gene (pMG168) were stable when maintained at low copy number. When the copy number of plasmid pMG168 was increased by putting replication under the control of the lambda PR promoter and the cI857 temperature sensitive repressor, expression of prochymosin was achieved. This strategy enables large-scale production of prochymosin without the need for antibiotic selection or other methods of preventing plasmid loss.
Chymosin↗
Dual-origin plasmid vectors whose origin of replication is controlled by the coliphage lambda promoter pL.
The insertion of an XhoI linker 30 bp from the 5' end of the RNA II primer for ColE1 plasmid replication has allowed us to replace the natural RNA II promoter by other controllable promoters, in particular lambda pL. Manipulation of this modified origin was facilitated by constructing dual-origin plasmids, stable maintenance being directed by the pSC101 origin. Experiments showed that such dual-origin plasmids could be stably maintained at approximately four copies per chromosome at 30 degrees C and readily amplified by thermal induction of strains carrying a thermolabile lambda repressor. The use of such plasmids for the construction of stable, amplifiable expression vectors is discussed.
Bacteriocin Plasmids↗
Cytotoxic T lymphocytes in DBA/2 mice harboring L5178Y cells in a tumor-dormant state.
Previous experiments have demonstrated a temporal relationship between the decline of cytotoxic T lymphocyte (CTL) activity in the peritoneal cavity of DBA/2 mice harboring L5178Y cells in a tumor-dormant state and the appearance of ascitic tumors. Some tumor-dormant mice remain clinically normal for many weeks after the decline of CTL activity, and this activity can be rapidly restimulated by an IP inoculation of irradiated L5178Y cells. We report here that the peritoneal cells from many tumor-dormant mice can be stimulated to cytolytic activity in vitro when cultured for 4 days either with or without the addition of irradiated L5178Y cells. Peritoneal cell populations which cannot be stimulated in vitro can suppress the generation of CTL in those populations which can be stimulated. The tumor-dormant state may terminate when suppressor cells in the peritoneal cavity of tumor-dormant mice inhibit the generation of CTL activity and permit tumor cells to produce an ascitic tumor.
Animals↗
Enhanced suppressor macrophage activity associated with termination of the L5178Y cell tumor-dormant state in DBA/2 mice.
Both cytolytic T-lymphocytes and cytolytic macrophages have been implicated in the long-term maintenance of L5178Y cells in a tumor-dormant state in DBA/2 mice. Eventually, however, the tumor-dormant state terminates, and all mice develop ascitic tumors. In an evaluation of the mechanisms involved in termination of the tumor-dormant state, we detected in the peritoneal cavity of many tumor-dormant mice macrophages with increased capacity to suppress the in vitro generation of a secondary anti-L5178Y cell cytolytic T-lymphocyte response. The incidence of macrophage-mediated immunosuppressive activity in individual tumor-dormant mice was related directly to the number of tumor cells in the peritoneal cavity of those mice. Furthermore, in tumor-dormant mice harboring fewer than 5 X 10(4) L5178Y cells, the detection of macrophage-mediated immunosuppressive activity was a prognostic indicator of termination of the tumor-dormant state and development of an ascitic tumor. These data suggest that peritoneal macrophage-mediated immunosuppressive activity, through inhibition of cytolytic T-lymphocyte generation in vivo, contributes to the termination of the tumor-dormant state and development of ascitic tumors.
Animals↗