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The stability of levofloxacin in 10 commonly used infusion fluids was studied. Levofloxacin 25-mg/mL injection was diluted to 0.5 and 5 mg/mL in each of the following i.v. infusion fluids: 0.9% sodium chloride injection, 5% dextrose injection, 5% dextrose and 0.9% sodium chloride injection, 5% dextrose and lactated Ringer's injection, 5% sodium bicarbonate injection, Plasma-Lyte 56 and 5% dextrose injection, 5% dextrose and 0.45% sodium chloride and 0.15% potassium chloride injection, 1/6 M sodium lactate injection, sterile water for injection, and 20% mannitol injection. Ten polyvinyl chloride bags were prepared for each solution; two were stored for 3 days at 25 degrees C, two for 7 days at 5 degrees C, two for 14 days at 5 degrees C, two for 13 weeks at -20 degrees C followed by 14 days at 5 degrees C, and two for 26 weeks at -20 degrees C, all in the dark. The solutions were visually examined, tested for turbidity and particulate matter, and subjected to stability-indicating high-performance liquid chromatography; solution pH was determined. Levofloxacin was stable in and compatible with all but two of the diluents tested. Precipitation occurred under all conditions in 20% mannitol injection for the 0.5-mg/mL levofloxacin concentration and after storage for 13 weeks at -20 degrees C for the 5-mg/mL concentration. Levofloxacin 0.5 mg/mL in 5% sodium bicarbonate injection formed a precipitate when stored at -20 degrees C for 13 weeks and beyond. Levofloxacin 0.5 and 5 mg/mL was compatible with and stable in 8 of the 10 infusion fluids studied.

The corneas of latently infected mice were UV irradiated to induce reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG). On days 1 to 4 after irradiation, TG were removed, serially sectioned and double stained to identify immune cells and virus antigens. Virus antigen was detected in small numbers (most commonly one) of neurons per ganglion as early as day 1, confirming the rapidity of reactivation and the neuron as the likely site of this event. The immune response was also rapid and effective since virus antigen was identified in immune cells at day 1 and by day 4 all samples were negative. The predominant infiltrating cells on days 1 and 2, when virus antigen was present and being cleared, were T cells, both CD4+ and CD8+. Later, large numbers of B cells appeared, suggesting that local antibody production may also be involved in controlling the reactivated infection. The observations suggest that a significant proportion of reactivation events do not result in disease of the eye or shedding of virus in the tear film. However, they also suggest that as little as one reactivating neuron in the ganglion may be sufficient to lead to such disease and/or shedding.

1. Isometric contractile characteristics of fast-twitch (flexor digitorum longus, FDL; medial gastrocnemius, MG) and slow-twitch (soleus) muscles were determined in pentobarbitone-anaesthetized fetal sheep between 90 and 140 days gestation. Five fetuses were hypophysectomized (HPX) at 90-95 days gestation and then studied at 138-140 days. 2. At 90-95 days gestation the time to peak of single twitch contractions for the soleus, MG and FDL were not significantly different from each other; the mean value (+/-S.E.M.) for all the muscles at this age was 77.6 +/- 9.0 ms. At 120-125 days gestation the MG and FDL contracted significantly faster (44.0 +/- 0.9 and 40.8 +/- 1.8 ms, respectively) than at 90-95 days, and did not change significantly thereafter. In contrast, the soleus muscle contracted more slowly (111.9 +/- 6.6 ms) at 138-140 days than at 90-95 days and 120-125 days gestation. 3. Soleus muscle consisted of type I fibres at all gestational ages. There was no significant change with gestational age in the relative numbers of type I and II fibres in the MG and FDL, but in the diaphragm the number of type I fibres increased and the number of type II fibres decreased between 125 and 138 days gestation. 4. HPX abolished the normal increase of soleus weight relative to body weight between 125 and 138 days but did not alter the change of twitch contraction time with age. HPX significantly prolonged twitch time to peak and time to half-relaxation of MG and time to half-relaxation of FDL at 138 days. 5. The maximum rate of rise of the isometric tetanic contraction was unchanged by HPX in all three hindlimb muscles, but fatigue of MG and FDL was increased. 6. The relative proportions of different fibre types in the hindlimb muscles and the diaphragm were unchanged by HPX, but there was a significant decrease in mean areas of type I and II fibres in the FDL and MG of the HPX fetuses.

Gangliosides are glycosphingolipids found ubiquitously on the surface of mammalian cells. They contain a ceramide tail that is inserted into the membrane and exposed carbohydrate and sialic acid moieties. The non-toxic B subunit oligomer (EtxB) of Escherichia coli heat-labile enterotoxin (Etx) is a potent immunogen in vivo and has profound modulatory effects on EtxB-primed lymphocytes in vitro, properties which are dependent on its ability to bind to GM1 ganglioside receptors. Here, it is shown that cross-linking GM1 by EtxB causes a differential effect on mature CD4(+) and CD8(+) T cells from lymph node cultures proliferating in response to an unrelated antigen, ovalbumin. Addition of EtxB to such cultures led to the complete depletion of CD8(+) T cells compared with enhanced activation of CD4(+) cells [as measured by expression of CD25 (IL-2Ralpha)]. By contrast, addition of a mutant EtxB, EtxB(G33D), which does not bind to GM1, failed to trigger CD8(+) T cell depletion. When EtxB was added to isolated non-immune CD8(+) lymphocytes rapid (12-18 h) alterations in nuclear morphology and the appearance of sub-G0/G1 levels of DNA were induced; properties which are characteristic of cells undergoing apoptosis. EtxB(G33D) failed to trigger apoptosis, indicating that the induction of the apoptotic signal was dependent on the binding of GM1. These findings provide an insight into the potent immunogenicity and immunomodulatory properties of E. coli enterotoxins as well as heralding a novel method for the selective induction of apoptosis in mature CD8(+) T lymphocytes.

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many of which were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon gamma. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

The ability of cells isolated from bovine milk and peripheral blood to present soluble protein and particulate bacterial antigens to peripheral blood T lymphocytes was compared using a culture system which consistently supports antigen-specific, primary, proliferative responses. The present study shows that cells from blood and from milk can present antigen to unprimed T cells. Major histocompatibility complex class II restriction of the responses was demonstrated by abrogation of proliferation by the addition of anti-bovine class II monoclonal antibody to cultures. Although cells derived from blood or milk were shown to be capable of presenting antigen to T cells, differences in optimal culture conditions and kinetics of the resulting response were observed.

We describe a method for immunohistochemical localization of T-cells, CD4+ T-cells, CD8+ T-cells, B-cells, activated lymphocytes, major histocompatibility complex (MHC) class II antigens, macrophages, dendritic cells, and granulocytes in rat and mouse tissue fixed in periodate-lysine-paraformaldehyde (PLP) and embedded in paraffin. Rat and mouse spleen and eyes were fixed in PLP for 18-24 hr, rapidly dehydrated, infiltrated under vacuum with paraffin at 54 degrees C, sectioned, and stained with appropriate monoclonal antibodies (MAbs). Sections of PLP-fixed, paraffin-embedded spleen were compared with acetone-fixed frozen spleen sections with respect to morphology and staining quality. Nine of 10 MAbs to rat antigens and eight of nine MAbs to mouse antigens stained paraffin sections equally or more intensely than frozen sections. The two MAbs that showed weaker staining still gave good staining on paraffin sections. Paraffin-embedded rat and mouse eyes were easier to section serially than frozen eyes, showed superior morphology, and individually stained cells were readily identified. Therefore, a combination of PLP fixation and low-temperature paraffin embedding permits detection of the major types of immune cell in rat and mouse tissues while maintaining good morphology, particularly in diseased, damaged, or delicate tissues.

The factors that may cause antigen-presenting cells to alter the pattern of protein processing and presentation to autoreactive T cells, and thereby stimulate autoimmune disease, are currently under debate. In this article, Chris Elson and colleagues suggest that cytokines associated with T helper 1 (Th1) cells alter the processing of proteins and that this effect can be counteracted by Th2-associated cytokines.

Pentamidine isethionate is a lyophilized antiprotozoal drug. Its solutions, when lyophilized, have been found to exhibit a range of properties that depend on its concentration and the manner in which it is allowed to freeze. These properties were examined by Differential Scanning Calorimetry (DSC), freeze-drying microscopy and X-ray powder diffraction. In DSC studies, the drug exhibited eutectic behavior with a eutectic temperature of -2.8 degrees C. Freeze-drying microscopic examination of a sample of solution after cooling at about 1 degrees C/min showed that the sample dried with retention up to -4 degrees C, where melting was observed. By using the appropriate drug concentration and manipulating the freezing stage, crystallization of the drug from solution could be induced during cooling to yield a crystalline end-product.

A self-report, attitudinal questionnaire was administered to 1,717 adult Australians between 18 and 40 years old to examine the effects of age and gender on psychological reactance. Analysis yielded a significant age effect: As age increased, the level of reactance tended to decrease. No significant differences in reactance emerged in relation to gender. A significant interaction between age and gender was found.

All vertebrates possess anatomical features not seen in their closest living relatives, the protochordates (tunicates and amphioxus). Some of these features depend on developmental processes or cellular behaviours that are again unique to vertebrates. We are interested in the genetic changes that may have permitted the origin of these innovations. Gene duplication, followed by functional divergence of new genes, may be one class of mutation that permits major evolutionary change. Here we examine the hypothesis that gene duplication events occurred close to the origin and early radiation of the vertebrates. Genome size comparisons are compatible with the occurrence of duplications close to vertebrate origins; more precise insight comes from cloning and phylogenetic analysis of gene families from amphioxus, tunicates and vertebrates. Comparisons of Hox gene clusters, other homeobox gene families, Wnt genes and insulin-related genes all indicate that there was a major phase of gene duplication close to vertebrate origins, after divergence from the amphioxus lineage; we suggest there was probably a second phase of duplication close to jawed vertebrate origins. From amphioxus and vertebrate homeobox gene expression patterns, we suggest that there are multiple routes by which new genes arising from gene duplication acquire new functions and permit the evolution of developmental innovations.

Reduction in subvisible particle counts in parenteral formulations is an important requirement. Despite selection of best container/closure and use of processing conditions where the drug is most soluble and stable, lyophilized formulations often demonstrate problems of subvisible particle counts greater than the permitted compendial limits. We have studied some formulation and processing approaches with different compounds with the intention of developing lyophilized products, which after reconstitution, pass compendial subvisible particle limits for large volume parenterals (LVP). The approaches studied were targeted to reduce "intrinsic" subvisible particle counts and included the addition of a solubilizing agent, pH adjustment, charcoal treatment of the process solution, and use of a filter with finer pore size for filtering the solution prior to lyophilization. Although the relative efficiency of each of these parameters in reducing subvisible particle counts was found to be slightly different for each compound, the inclusion of a solubilizing agent and charcoal treatment of the processing solution were found to have the most pronounced effect. The combination of two or more parameters was often found to be even more effective. It is suggested that these approaches may be useful in developing other lyophilized products that yield low subvisible particle counts on reconstitution.

The mechanical properties of frozen mannitol, L-iditol, dulcitol, and sorbitol solutions were measured as a function of temperature during warming (after freezing) using a thermal mechanical analyzer (TMA). The mannitol sample first underwent a contractive phase starting at 30 degrees C and ending at -25 degrees C. This was followed by an initially rapid, then less rapid expansive phase which continued until melting occurred. These events provide direct evidence that mannitol solutions undergo an expansion during warming after "fast" freezing and also corroborate the vial breakage observed when mannitol solutions are frozen and thawed or freeze-dried in glass vials. Of the other stereoisomers studied, only dulcitol had an expansive phase although at a slower rate than mannitol. Its solution was found to cause vial breakage also but only at fill volumes higher than mannitol fill volumes. TMA is a useful technique for studying dimensional changes in the frozen state, with important implications for solving vial breakage problems in freeze-drying.

We have studied the ability of mononuclear cells extracted from the colon (CMC), draining lymph nodes (LNMC), peripheral blood (PBMC) and spleen (SMC) of a patient with ulcerative colitis and severe autoimmune haemolytic anemia to produce red cell autoantibodies. The LNMC, PBMC and SMC secreted immunoglobulin in vitro in pokeweed mitogen culture but not spontaneously. They also produced immunoglobulin when transferred to severe combined immunodeficient (SCID) mice but no anti-red cell activity was demonstrable. In contrast, in vitro cultures of CMC produced immunoglobulin spontaneously, showing the presence of activated B-cells and plasma cells, but anti-red cell activity could not be demonstrated. However, CMC transferred to SCID mice were able to produce IgG with anti-red cell activity. These results concur with clinical observations suggesting that the colon is the source of red cell autoantibodies in these patients.

As in HIV infection of humans, cats infected with FIV are particularly susceptible to secondary infection by opportunistic pathogens, suggesting an impaired ability to elicit an effective immune response against foreign antigens. In order to investigate the development of immunity in FIV-infected cats, we have used an autologous culture system to directly measure priming of naive CD4+ T cells to soluble protein antigen, in vitro. Using this assay, we showed previously that cats infected with FIV for several months had significantly reduced primary proliferative responses. We have now examined cats before infection, and at varying times after infection with FIV, to determine how soon after infection this defect in T cell priming was evident, compared with other quantitative and qualitative measurements of lymphocyte function. Our results showed a progressive decline in immune function in asymptomatic cats during the acute stage of infection with FIV. Primary T cell responses were most sensitive and a significant reduction in proliferation of naive T cells to foreign antigen occurred 5 weeks after infection, despite normal blastogenesis to T cell mitogens and normal CD4+/CD8+ ratios at these times. Whilst lymphocyte proliferation to T cell mitogens was unaffected throughout, a significant reduction in proliferation to a B cell mitogen occurred from week 8 onwards. CD4+/CD8+ ratios fell significantly from week 13 onwards, and proliferation of the memory T cell population to a recall antigen was significantly impaired later, from week 19 onwards. The defect in the priming of naive T cells to foreign antigen early after infection may be important in determining susceptibility to secondary infections.

Spleen cells, but not mesenteric lymph node cells, from 3-week-old piglets abruptly weaned onto a soya-based diet, produced less interleukin-2 (IL-2) following non-specific activation with concanavalin A (Con A) than did cells from age- and litter-matched, unweaned controls. In contrast, the ability to express receptors for IL-2 was only marginally reduced. The effect on IL-2 production was most marked in animals weaned for as little as 24-48 h. Variation within groups increased with time after weaning, indicating differences between individuals in the longer-term effects of weaning. This finding may be due to endogenous production of steroids resulting in generalised impaired immune function or to retention of cells within intestinal sites owing to an active local immune response.

The embryology of amphioxus has much in common with vertebrate embryology, reflecting a close phylogenetic relationship between the two groups. Amphioxus embryology is simpler in several key respects, however, including a lack of pronounced craniofacial morphogenesis. To gain an insight into the molecular changes that accompanied the evolution of vertebrate embryology, and into the relationship between the amphioxus and vertebrate body plans, we have undertaken the first molecular level investigation of amphioxus embryonic development. We report the cloning, complete DNA sequence determination, sequence analysis and expression analysis of an amphioxus homeobox gene, AmphiHox3, evolutionarily homologous to the third-most 3' paralogous group of mammalian Hox genes. Sequence comparison to a mammalian homologue, mouse Hox-2.7 (HoxB3), reveals several stretches of amino acid conservation within the deduced protein sequences. Whole mount in situ hybridization reveals localized expression of AmphiHox3 in the posterior mesoderm (but not in the somites), and region-specific expression in the dorsal nerve cord, of amphioxus neurulae, later embryos and larvae. The anterior limit to expression in the nerve cord is at the level of the four/five somite boundary at the neurula stage, and stabilises to just anterior to the first nerve cord pigment spot to form. Comparison to the anterior expression boundary of mouse Hox-2.7 (HoxB3) and related genes suggests that the vertebrate brain is homologous to an extensive region of the amphioxus nerve cord that contains the cerebral vesicle (a region at the extreme rostral tip) and extends posterior to somite four.(ABSTRACT TRUNCATED AT 250 WORDS)

PROBIT estimates biologic activity by weighted probit regression analysis and comparison with a known standard. The program is intended to speed up calculation of the concentration of cytokines in large numbers of cell supernatants. Data are input from either sequential or free-form text files created with a spreadsheet or text editor. This allows transfer of data from a beta-counter equipped with a suitable terminal without retyping. Results are displayed and/or printed as relative potency (% of standard) and 50% effective dose (ED50).

To understand the local immune events which occur when a novel antigen is encountered in the gut it is necessary to know whether cells from the mucosal tissues are capable of initiating T-cell reactivity. We have examined the capacity of cells isolated from the Peyer's patches and the lamina propria of the murine small intestine to present keyhole limpet haemocyanin (KLH) to naive syngeneic splenic T cells in vitro. The properties of the gut antigen-presenting cells (APC) were compared with those of cells from the spleen and the mesenteric lymph nodes. Results clearly demonstrate that cells from the lamina propria as well as from the Peyer's patches, mesenteric lymph node and spleen can present KLH to naive T cells, inducing strong proliferative reactions comparable in magnitude and kinetics. All the APC populations tested induced interleukin-2 (IL-2) production in primary cultures, although minor differences were noted when lamina propria cells were used as APC. IL-4 was not detected in supernatants from cultures of non-immune T cells in the presence of APC from any tissue. Phenotypic analysis of the cells in cultures of naive T cells, with antigen and APC from different gut-associated tissues revealed important differences. Cells from Peyer's patch, mesenteric lymph node and spleen gave rise to cultures containing largely CD4+CD8- cells. However, cultures in which lamina propria cells acted as APC consisted primarily of CD4-CD8+ cells.

Objective: Cats naturally infected with feline immunodeficiency virus (FIV) are particularly susceptible to infection with opportunistic pathogens, suggesting that these animals are unable to develop an effective immune response against the pathogen. Previous studies have used CD4+:CD8+ lymphocyte ratios and mitogen blastogenesis to identify immunological abnormalities in FIV-infected cats. However, these studies provide limited information for understanding the nature of the cellular dysfunction in FIV-infected cats, particularly defects in antigen-specific immune responses., Design: To investigate whether cats infected with FIV are less able to mount an immune response to previously unencountered antigens, we compared the development of antigen-specific cellular immunity at the stage of T-cell priming in uninfected and FIV-infected cats., Interventions: The general immune status of cats was assessed by peripheral blood CD4+:CD8+ lymphocyte ratios (flow cytometry), and by lymphocyte blastogenesis response to T- and B-cell mitogens. In addition, we describe the development of an autologous culture system to measure specific priming of naive feline T-cells to soluble antigen in vitro. This assay was used to compare T-cell priming in uninfected cats and cats which had been infected with FIV for 6-27 months., Results: As in HIV infection, CD4+:CD8+ lymphocyte ratios in FIV-infected cats were found to be inverted, due to a reduction in the percentage of CD4+ cells. In addition, lymphocyte blastogenesis to both T- and B-cell mitogens was significantly impaired in FIV-infected cats. Priming to keyhole limpet haemocyanin (KLH) elicited a late proliferative response resulting from the expansion of CD4+ (T-helper cells). T-cell growth factor secretion correlated with cell proliferation. Restimulation of cells with fresh antigen-presenting cells and antigen showed that antigen-specific T-cell priming had occurred in the initial culture. When primary proliferation responses in FIV-infected cats were examined, it was observed that naive CD4+ T-cells from FIV-infected cats were significantly impaired (P less than 0.001) in their ability to be primed to KLH when compared with uninfected controls., Conclusions: Impaired priming of naive CD4+ T-helper cells to antigen in FIV-infected cats may explain the increased susceptibility of these animals to infection by opportunistic pathogens. The poor ability of human patients with AIDS to develop humoral immunity following vaccination may also be caused by such a defect in T-cell priming.

A number of agents have been shown to alter the latent state of herpes simplex virus in murine sensory ganglia. However, it seems that effective triggers of recrudescent disease must act not only to reactivate latent HSV infection, but also to create a favorable environment in the skin for viral replication. The possibility that alteration of the local Langerhans cell population is one way in which effective triggers of recrudescence may act has been investigated. Of the agents tested, which affect latent HSV, only DMSO significantly altered the numbers of ATPase-bearing Langerhans cells in the epidermis, maximally reducing their density by 83% in 48 h. Xylene and retinoic acid had no discernible effect on numbers of ATPase-staining cells over the 4 d tested. However, the extent to which agents reduced ATPase-staining cell numbers did not correlate with their ability to affect the antigen-presenting capacity of the cells in HSV-specific T-cell proliferative assays in vitro. Xylene and retinoic acid markedly reduced the accessory cell function of epidermal cell suspensions, whereas DMSO had no effect.

We have used the polymerase chain reaction (PCR) to amplify, clone and sequence homologues of the Drosophila segment polarity genes engrailed (en), cubitus interruptus Dominant (ciD) and wingless (wg) from the genome of the brachiopod, Terebratulina retusa (Linnaeus). The deduced translation products of brachiopod en and ciD share high levels of sequence identity with their Drosophila homologues. The brachiopod wg-related clone is divergent from Drosophila wg, although clearly a member of the wg/Wnt gene family. These results indicate that structural diversity of Drosophila segment polarity genes has been evolutionarily conserved in a divergent, ancient and unsegmented animal phylum.

The in vitro production of a variety of cytokines by lymphocytes isolated from spleen mesenteric lymph node (MLN), Peyer's patches (PP) and lamina propria (LP) was measured, after oral immunization with keyhole limpet hemocyanin using cholera toxin as an adjuvant. LP responses were characterized by very high levels of interleukin (IL) 4, IL 5 and IL 6 with lower levels of IL 2 and interferon-gamma (IFN-gamma). The PP had lower levels of IL 4, IL 5 and IL 6 than LP but higher levels of IL 2, IFN-gamma was only present at very low levels in this organ. The MLN had a pattern of cytokine production similar to the PP but did produce IFN-gamma. The spleen produced all cytokines measured except IL 5. Antibody production was characterized by IgA in the LP and PP but IgG was the dominant isotype in the spleen, the MLN was a poor source of antibody-producing cells. We interpret the results to show that (a) the LP response to cholera toxin/keyhole limpet hemocyanin is dominated by Th2-type cytokines compared to a lower production of Th1 type and (b) that the PP has responses typical of an organ with a high proportion of resting lymphocytes which develop mainly into Th2-types cells. The spleen is less dominated by Th2-type cytokines than the mucosal sites and this difference is paralleled by IgA antibody production at the mucosal sites and IgG antibody dominance in the spleen.

Traditional approaches to phylogeny reconstruction have not allowed precise resolution of the evolutionary relationships between the major deuterostome phyla (chordates, hemichordates, echinoderms). Here we report the use of a molecular approach to investigate deuterostome phylogeny. We have used a polymerase chain reaction-based strategy to amplify, clone and sequence parts of the genes coding for 18S ribosomal RNA from Saccoglossus cambrensis (Hemichordata), Arbacia sp. (Echinodermata) and, for comparison, Mytilus edulis (Mollusca). We report the results of phylogenetic reconstructions using these, and homologous sequences from other eukaryotes. The results of our analyses are consistent with the hypothesis that S. cambrensis and vertebrates share a common ancestor not shared by echinoderms.

Cholera toxin is widely recognized as a potent stimulator of mucosal IgA responses after oral feeding. However, comparatively little is known of its ability to stimulate cellular responses. This is due in part to the direct inhibitory effects of cholera toxin on T lymphocytes, which can confound attempts to study primed T-cell responses in vitro. We have avoided this problem by using cholera toxin as an adjuvant to enhance the response to an unrelated protein fed simultaneously. Thus the simultaneous feeding of 10 micrograms of cholera toxin and 5 mg of keyhole limpet haemocyanin (KLH) results in the priming of T cells in the spleen, mesenteric lymph nodes, Peyer's patch and lamina propria that proliferate when restimulated with KLH in vitro. The feeding of KLH alone does not result in such responses. Both CD4- and CD8-positive antigen-specific T cells were involved in the response and the cells produced both interleukins 2 and 4 on antigen restimulation in vitro.

The thermal characteristics as a function of cooling rate of mannitol solutions were studied by Differential Scanning Calorimetry (DSC). The exotherm appearing during warming at -25 +/- 1 degree C could be correlated with the temperature at which frozen mannitol solutions broke vials during warming. This temperature was also consistent with the temperature at which a sharp increase in resistance was reported in a preceding paper. The DSC exotherm did not appear to be due to crystallization of amorphous mannitol but rather to some other type of solid state transition. The peak was initially broadened and then obliterated by increasing amounts of additives such as lactose, glycine, potassium chloride, and sodium chloride. The presence of these ingredients in small amounts (less than 1% of the mannitol concentration) significantly reduced or prevented breakage. Other stereoisomers of mannitol, e.g., sorbitol and dulcitol, did not show any exotherm or break vials on freezing and thawing. It was also found that different types of vials exhibited different susceptibilities to breakage.

The role of epidermal Langerhans' cells in infection with herpes simplex virus (HSV) was investigated using a culture system that supports antigen-specific primary and secondary T-cell proliferative responses. Epidermal cell suspensions were capable of restimulating the response of in vivo primed T cells to UV-inactivated HSV. This capability was also present in cell suspensions enriched for Langerhans' cells, but was abrogated by the depletion of I-A-bearing cells. The magnitude, kinetics and phenotype of the responding cells were similar to those elicited when HSV was presented to primed T cells by antigen-presenting cells from the spleen. In marked contrast, whereas splenic antigen-presenting cells induced strong antigen-specific proliferation of unprimed T cells (primarily of the helper phenotype), Langerhans' cells failed to invoke any detectable reaction of such cells.

The Drosophila melanogaster developmental gene engrailed (en) is a member of a distinct subfamily of homeobox genes with a wide phylogenetic distribution. Here we report the use of reduced stringency polymerase chain reaction (PCR) to amplify and clone 8 genes related to en from 5 vertebrate species, including representatives of the most ancient vertebrate lineages. Nucleotide and deduced amino acid sequence comparisons between mouse, toad, zebrafish, lamprey and hagfish genes reveal extensive evolutionary conservation, and suggests that 2 en-like genes have been retained in most vertebrate lineages.

The capacity of epidermal cells (EC) to present antigen to primed and non-immune T cells was investigated using a culture system that supports antigen-specific primary and secondary proliferative responses. Although both naive and bovine serum albumin (BSA)-immune T cells reacted against BSA in the presence of either splenic or epidermal antigen-presenting cells (APC), important differences were noted in the kinetics and the magnitudes of the various responses. Most conspicuous was the relatively poor primary response supported by EC which evidently elicited very few BSA-immune T-helper cells. Despite this, the primed antigen-specific T cells recovered were phenotypically similar to those resulting from the stronger primary responses induced by spleen cells. In contrast to this disparity in the ability to prime, EC and spleen cells stimulated secondary reactions of comparable magnitude. We therefore consider that, in comparison with splenic APC, EC may require some additional stimulus to acquire the capacity to prime.

One thousand and seventy-six birds of 26 families and 127 species were examined for hemoprotozoa; 404 birds (37%) of 41 species representing 17 families harbored one or more blood parasites. Most parasites were species of Haemoproteus which represented 95% of all parasitic infections detected. Prevalence of blood parasites in birds collected from four areas over a period of six years was relatively stable.

The type, paratype, and reference material of avian hematozoa in the reference collection of the International Reference Centre for Avian Haematozoa is listed. This material includes one species of Atoxoplasma, 41 species of Haemoproteus, one species of Hepatozoon, two species of Lankesterella, 15 species of Leucocytozoon, 31 species of Plamodium, one species of Spirogregarina, and three species of Trypanosoma.

The use of the reduced three-suffix solubility equation in characterizing solubility in ethanol-water mixtures is discussed. The equation states that ln xs2,m = z1 ln xs2,1 + z3 ln xs2,3 - A1-3z1z3(2z1 - 1)- (q2/q1) + A3-12z21z3(q2/q3) + C2z1z3, where xs2,1, xs2,3, and xs2,m are the mole fraction solubilities of the solute in ethanol (subscript 1), water (subscript 3), and in the mixture (m); A1-3 and A3-1 are solvent-solvent interaction terms; C2 is a solute-solvent interaction term; and the q- and z-values are molar volumes and solute-free volume fractions, respectively. The contributions of the various terms in the equation to solubility are examined, and the possible use of its derivative in indicating whether a maximum may exist in the solubility profile is discussed. Methods of obtaining the solvent-solvent interaction constant and the ternary constant C2 are described, and the general effectiveness of the equation in describing solubility is examined. The equation is shown to be applicable to 10 compounds with widely different physical properties and, thus, appears to combine both ease of use and general utility.

A total of 389 birds of 32 species representing 14 families from Tchad were examined for blood parasites. Eighty-nine (22.9%) harbored infections of Haemoproteus (64%), Plasmodium (12.4%), Trypanosoma (1.1%), Atoxoplasma (=Lankesterella) (5.6%), and microfilaria (28.1%). Species of Leucocytozoon were not observed in the present study. The occurrence of the different genera differed markedly between bird families; members of the Ploceidae comprised 85% of the infected birds.

An approach is developed by which the solubility of an organic compound in mixed solvents may be estimated. In this approach, an expression for the excess Gibbs free energy of mixing for multicomponent solvent systems was used to obtain parameters characteristic of the interaction between the solvents. A fairly simple equation which predicts the solubility of a solute in a binary solvent system over the entire solvent composition range was then derived. The equation may be partitioned into terms that contain (a) pure solvent solubilities, (b) solvent-solvent interaction contributions, and (c) contributions from the solute-mixed solvent interactions. The required data are the molar volume of the solute, the pure solvent solubilities, and, theoretically, one experimentally determined solubility in a solvent mixture. The equation can be easily extended for systems with three or more solvents.

Blood films from 60 mallard (Anas platyrhynchos) and 67 pintail (A. acuta) ducks, collected in Alberta and the Mackenzie Delta, Northwest Territories, during 1973 and 1974, were examined for blood parasites. Twenty-two (37%) of the mallards and fourteen (21%) of the pintails were infected with one or more species of hematozoa. Infections of Leucocytozon simondi occurred more frequently (86%) than Haemoproteus nettionis (22%) in the infected birds. Trypanosoma avium occurred in one individual of each species of duck; one pintail harbored an unidentified microfilaria. Differences of prevalence between species are predicted on the basis of host attractancy to vectors and/or host habitat selection, and are discussed.

The reduced three-suffix solubility equation derived from the Wohl excess free energy expression is used to describe the solubility of phenobarbital in propylene glycol-water, ethanol-propylene glycol, and ethanol-water-propylene glycol mixtures and the solubility of hydrocortisone in propylene glycol-water mixtures. Solvent-solvent interaction constants were obtained by fitting total vapor pressure versus composition data, obtained at 25 +/- 0.1 degrees C, to the Wohl excess free energy model for the solvents. The equation describes solubility in these systems satisfactorily except for phenobarbital in ethanol-propylene glycol, where the solubility is fairly high and assumptions involved in the derivation of the equation do not hold.

The 1983 National Institute of Mental Health (NIMH) guidelines for a premenstrual syndrome (PMS) diagnosis were applied to Daily Rating Form (DRF) data in a prospective research study. The use of two sets of criteria, each consistent with the NIMH guidelines, resulted in prevalence rates of 44% and 17%. The authors discuss other problems in defining a "syndrome": symptom patterns and the relationship between PMS and the presence of a psychiatric disorder.

This study investigated the relationships of positive and negative life change to yeast infections in women having a gynecological examination at a university health center. Subjects completed the Life Experiences Survey and a questionnaire about experiences with yeast infections and received, as a routine part of their visitation of the gynecology service, a standard gynecological examination, including a laboratory test for yeast infections. Positive life change was unrelated to any of the variables regarding yeast infections and was only minimally correlated with negative life change. Negative life change, while not related to the presence of current yeast infections, was positively correlated with the reported number of yeast infections during the past year, concern about these infections, and a number of physician visits for yeast infections. Negative life change was also negatively correlated with grade point average. Neither antibiotics nor contraceptive pill use interacted with negative life change to influence experiences with yeast infections.

A total of 809 birds from Senegal, including 43 species and 21 families, were examined for hematozoans; 93 birds (11.5%) harbored blood parasites, with only 7 (7.5%) harboring mixed infections. Species of Haemoproteus occurred in 81.7% of the infected birds while species of Plasmodium. Trypanosoma, microfilaria and Leucocytozoon were encountered less frequently. The majority of the sample was composed of species of ploceids and estrildidis and blood parasites were most prevalent in the colonial-nesting ploceids. Prevalence of blood parasites in Senegal was low in comparison to that seen in birds from other parts of Africa.

The reduced 3-suffix solubility equation (R3SSE) is applied to the characterization of solubility in the ethanol-water system. The data needed are the solubility of the compound in each of the pure solvents and at one ethanol-water composition. This composition has been estimated from solubility data to be 0.56 volume fraction of ethanol. The solubility obtained at this volume fraction is used to estimate the ternary solute-solvent interaction constant, C2. The R3SSE, with the C2 thus obtained, predicts the mixed solvent solubilities of the compounds tested, as accurately as that obtained from several volume fractions. The superiority of the R3SSE over two related equations--a simple second-degree polynomial equation and a simplified form of the R3SSE which neglects contributions to solubility from the solvent mixture--is also demonstrated for a number of solutes.

In contrast to oral administration, topical ciclosporin is not effective in the treatment of psoriasis. This may be due to the drug's inability to penetrate the stratum corneum or to a need for systemic metabolism of ciclosporin to active metabolites. We conducted a preliminary evaluation of the ability of ciclosporin, in a variety of vehicles, to penetrate excised human skin in a two-compartment diffusion cell, a standard in vitro technique for evaluating percutaneous drug delivery. A high-pressure liquid chromatography assay for ciclosporin did not detect the passage of ciclosporin through skin. This finding was consistent through multiple trials, some of which evaluated Azone or liposomal formulations, which are reported to enhance the penetrability of certain compounds. These results indicate that ciclosporin is unable to penetrate human skin in the vehicles tested. This may explain the lack of clinical efficacy we demonstrated in 5 patients with psoriasis treated with topical ciclosporin for 14-16 days.