Endogenous prostaglandin E2 modulates calcium-induced differentiation in human skin keratinocytes.

The concentration of extracellular calcium appears critical to the initiation of keratinocyte differentiation. Prostaglandins (PGs) have also been implicated in cell differentiation. Consequently, the participation of endogenous eicosanoids in calcium-induced differentiation of human keratinocytes was evaluated in vitro. Our results demonstrate that: (1) exogenously introduced PGE2, the major keratinocyte-derived eicosanoid, but not prostaglandin I2 (PGI2) or its stable metabolite 6-keto-PGF1 alpha, enhances calcium-induced cornified envelope formation, an established marker of keratinocyte differentiation; (2) increasing extracellular calcium increased endogenous PGE2 synthesis by cultured keratinocytes; (3) blocking endogenous PGE2 synthesis with indomethacin significantly suppresses calcium-induced formation of the cornified envelope; and (4) adding back PGE2 to indomethacin-treated keratinocytes is able to re-establish the control level of cornified envelope formation following stimulation by calcium. These data document the participation of endogenously generated PGE2 in the modulation of calcium-induced differentiation by human keratinocytes.

Stability of temperature asymmetries in reflex sympathetic dystrophy over time and changes in pain.

OBJECTIVE: To determine the clinical usefulness of skin temperature patterns for tracking reflex sympathetic dystrophy (RSD) by assessing (a) long-term relationships between changes in pain due to RSD and patterns of near surface blood flow and (b) relationships between site of pain and site of greatest asymmetries in near surface blood flow patterns. DESIGN: Multiple videothermographic evaluations of near surface blood flow patterns were performed on subjects diagnosed as having RSD. At each session, subjects filled in an outline of the body to show the location, intensity, and description of their pain. The thermograms were evaluated independently by two raters for location and intensity of pain, as well as location and degree of temperature asymmetries. SETTING: Two Army Medical Centers. SUBJECTS: Thirteen male and 16 female subjects were subsequently diagnosed as having RSD. OUTCOME MEASURES: Ratings of pain and videothermograms of the lower limbs were used as outcome measures. RESULTS: All but one subject were usually cooler on the most painful side by at least 0.5 degrees C. The amount of relative coolness was not proportional to pain intensity. There were no consistent overlaps between exact location of pain and greatest thermal asymmetry. Seven subjects were thermally symmetrical on at least one recording. Six subjects were warmer on the affected side on at least one recording. One subject was always warmer on the affected side. CONCLUSIONS: Videothermography is not an appropriate tool to use alone for either single session diagnosis or multi-session tracking of RSD.

Mecamylamine blockade of nicotine responses: evidence for two brain nicotinic receptors.

Mice of two inbred strains, DBA and C3H, were pretreated with mecamylamine before challenge with nicotine. Mecamylamine blocked nicotine-induced seizures, enhanced startle, and alterations in respiratory rate, Y-maze activity, heart rate and body temperature. Mecamylamine blocked nicotine-induced seizures and enhanced startle with IC50 values of less than 0.1 mg/kg. The other nicotine effects were blocked by mecamylamine with IC50 values between 0.8 and 2.3 mg/kg. Strain differences in sensitivity to mecamylamine blockade were also detected. These results suggest that nicotine elicits its effects at two receptors, which may be those labeled with [125I]-alpha-bungarotoxin and with [3H]-nicotine.

Temporal relationships between changes in phantom limb pain intensity and changes in surface electromyogram of the residual limb.

Previous studies of relationships between surface EMG of the residual limb and phantom pain have not shown which changed first. Thus, predictive relationships could not be demonstrated. 24 male (20) and female (4) amputees between the ages of 33 and 71 who reported either burning (3), cramping (8), shocking-shooting-stabbing (6), or a combination of these descriptions of phantom pain (7) participated in one or two recording sessions. Raw surface EMG from the major muscles of the residual limb was recorded while subjects activated an event marker to indicate changes in pain. All eight subjects with cramping phantom pain reported changes in pain after the recording showed sharply demarcated increases in EMG. Subjects reporting either shocking-shooting or burning pain did not show any consistent relationships between EMG and pain. Three of the four subjects reporting experiencing both shocking-shooting and cramping phantom pain simultaneously during recordings showed changes in EMG preceding changes in pain. Sensations of cramping phantom pain were preceded by increases in muscle tension in the residual limb in almost every instance for each of our subjects showing changes in cramping phantom pain. Thus, changes in muscle tension in the residual limb are likely to either be causes or close intermediaries for the cause of cramping phantom pain but not necessarily of other common descriptors.

Patient satisfaction with a levonorgestrel-releasing contraceptive implant. Reasons for and patterns of removal.

OBJECTIVE: To investigate the experience of the users of a levonorgestrel-releasing contraceptive implant (Nor-plant) and reasons for early removal. STUDY DESIGN: Two hundred fifty-one women who had Norplant placed through the Ohio State University Department of Obstetrics and Gynecology were sent a questionnaire that included general demographic questions and questions regarding preplacement education; reasons for choosing Norplant; problems experienced due to Norplant; reasons for removal, if applicable; and overall satisfaction. RESULTS: One hundred eleven questionnaires (44%) were completed and analyzed. Twenty-four (21.6%) of patients had Norplant removed, 32 (28.8%) planned on early removal, and 55 (49.5%) desired continued use. Ninety-eight percent of women who planned continued use of Norplant had received reading material as part of their preplacement counseling and 95% felt adequately counseled. This is significantly higher than women who had Norplant removed or planned on early removal. Convenience, problems with other contraceptive methods and effectiveness were the most important reasons why patients chose Norplant. Menstrual changes were the most common side effect, experienced by 80% of all women in the study. Weight gain, headache, mood changes, mastalgia and acne were also frequently reported. Women who had Norplant removed were significantly more likely to have experienced dizziness when compared to women desiring continued use. Women planning early removal were also more likely to have experienced dizziness in addition to acne, headache, hair loss and other side effects. Menstrual irregularities, followed by mood changes and headaches, were the most important side effects associated with early Norplant removal. Overall, 68% of users were at least somewhat satisfied with Norplant. CONCLUSION: Norplant contraception was a satisfactory form of birth control for the majority of patients in the study despite the frequent occurrence of side effects. Reading material regarding benefits and side effects of Norplant is an important aspect of patient counseling and may improve long-term use of Norplant.

PIP: To improve counseling services for new users of the levonorgestrel-releasing contraceptive implant (Norplant), the 251 women who had the implant inserted through the Ohio State University Department of Obstetrics and Gynecology during 1991-94 were mailed a questionnaire concerning their experiences. Responses were received from 111 (44%) of these women; 24 (21.6%) had already had the implants removed after a mean duration of use of 14.6 months, 32 (28.8%) planned on early removal, and 55 (49.5%) desired continued use. There were no significant differences between these 3 groups in terms of age, gravidity, parity, type of provider, or interval between counseling and placement. Among women who planned to continue Norplant use, 98% had been provided with reading material as part of their preinsertion counseling and 95% characterized this counseling as adequate; these rates were significantly lower in the other 2 groups. Frequently reported side effects included menstrual changes (80%), weight gain (53%), headache (50%), mood changes (59%), mastalgia (47%), and acne (47%). Menstrual irregularities, mood changes, and headaches were the side effects cited most frequently among women who opted for early removal. Overall, 75 users (68%) were satisfied or very satisfied with the method, 15 (14%) were somewhat dissatisfied, and 18 (16%) were very dissatisfied. Consistent distribution during preinsertion counseling of reading materials on Norplant and its side effects is recommended to enhance method acceptance.

Costing services: comparing three i.v. medication systems.

Three methods of intermittent intravenous (IV) medication delivery were studied for evidence of cost effectiveness. Eighty-five patients receiving antibiotics on two general medicine units were examined over an eight-week period. Chi square analysis revealed no significant differences in the number of IV complications among the three delivery systems. ANOVA revealed no significant differences among the three systems. A cost savings of $99.70 per patient using the Bard system and $98.60 using the BD system was realized. Thirty-six (88%) of the nurses preferred a syringe pump system, 10 patients showed no strong preference for any of the methods, and the two pharmacists preferred a syringe delivery system.

12-O-tetradecanoylphorbol-13-acetate and the induction of prostaglandin E2 generation by human keratinocytes: a re-evaluation.

12-O-Tetradecanoylphorbol-13-acetate (TPA) induces prostaglandin E2 (PGE2) synthesis in mouse keratinocytes and is associated with the induction of keratinocyte proliferation as well as accelerated differentiation. In human keratinocytes, TPA has been reported not to induce the release of either 3H-labeled arachidonic acid or 3H-labeled prostaglandins, even though cell differentiation is stimulated. Because PGE2 has been associated with the modulation of cell differentiation and because of technical problems inherent in evaluating arachidonic acid metabolism using only radiolabeled substrates, we evaluated the ability of TPA to induce endogenous PGE2 generation by cultured human keratinocytes using a specific and sensitive enzyme immunoassay. With this technique, TPA was found to induce a dose-dependent (1.6 x 10(-12)-1.6 x 10(-8) M) increase in PGE2 generation. These results are consistent with observations made not only in mouse keratinocytes but in other mammalian and human cell types. Documenting the ability of TPA to stimulate PGE2 production in human keratinocytes is very relevant to current theories regarding the role of PGE2 in keratinocyte differentiation as well as to establishing parallels between the murine and human skin models.

Resistance of mice suppressed for IgM production to Babesia microti infection.

The immunological mechanisms responsible for overcoming infections with Babesia, an intra-erythrocytic protozoan, are not fully understood. Although high titres of specific anti-babesial antibodies have been observed in several species of animals, and protection has been obtained by transfer of large volumes of recovery serum, the role of antibody in the immune response to an infection is uncertain. The present study investigates the nature of B-cell participation during Babesia microti infections by observing the course of the disease in mice in which IgM production has been suppressed from birth and which contain no B cells. The results show that, in contrast to control mice, which develop and subsequently clear circulating parasitaemias, suppressed mice show an unexpected resistance to infection as reflected by a virtual absence of parasites in the peripheral circulation.

Identification of Plasmodium falciparum histidine-rich protein 2 in the plasma of humans with malaria.

Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) is a water-soluble protein released from parasitized erythrocytes into in vitro culture supernatants. This study sought to determine whether PfHRP-2 could be detected in the plasma of humans with P. falciparum malaria. A monoclonal antibody (1E1) is described that binds to PfHRP-2. By using monoclonal antibody 1E1, PfHRP-2 was identified by Western blot (immunoblot) analysis in the plasma of 37 of 39 (95%) patients experiencing either a first or repeat episodes of P. falciparum malaria and by dot blot analysis in the plasma of 40 of 41 patients tested. PfHRP-2 was not detected in 30 control, uninfected subjects. The current demonstration of PfHRP-2 in plasma, plus the fact that it is a structurally well-characterized molecule present in all natural isolates of P. falciparum tested, makes PfHRP-2 of interest for its potential effects on the host immune system and as an antigen for specific diagnosis of malaria.

Antimalarial antibodies of the immunoglobulin G2a isotype modulate parasitemias in mice infected with Plasmodium yoelii.

Previous studies have demonstrated the importance of antibodies in mediating immunity to malaria, but the relative contribution of the different immunoglobulin isotypes has not been assessed. In this study, hyperimmune plasma was generated against Plasmodium yoelii and separated by protein A-Sepharose chromatography into fractions containing immunoglobulin G1 (IgG1), IgG2a, IgG2b, or IgG3 antibodies and the remaining nonbinding plasma proteins, including IgM. Following concentration, the antimalarial titer of each isotypic fraction was approximately equivalent to the corresponding isotype in hyperimmune plasma. The isotypic fractions were passively transferred to BALB/c and outbred ICR mice prior to challenge with virulent P. yoelii 17XL and to CBA/CaJ mice challenged with avirulent P. yoelii 17XNL. Only mice receiving IgG2a antibodies experienced an altered course of infection. Immunoprecipitation studies showed that all four IgG isotypes appear to recognize a similar set of antigens. These results suggest that antimalarial antibodies of the IgG2a isotype play a dominant role in modulating P. yoelii parasitemias.

Immunity to Plasmodium Berghei yoelii in mice. I. The course of infection in T cell and B cell deficient mice.

The course of infection with 17X nonlethal Plasmodium berghei yoelii was examined in BALB/c mice which were deficient in either T cells or B cells. Markedly increased parasitemia and mortality were observed in athymic (nude) mice which had been backcrossed on a BALB/c background (T cell deficient) compared to similar mice which had been grafted with neonatal BALB/c thymus, and were also observed in BALB/c mice suppressed from birth with goat antiserum to mouse mu-chain (B cell deficient) compared to age- and sex-matched BALB/c controls. These results establish the requirement for the presence of both T cells and B cells for effective resistance to an intercurrent infection with 17XNL P.b. yoelii in adult BALB/c mice. Mechanisms by which the requirement for both T cells and B cells could be explained were discussed. The model of mu suppression was shown to be a valuable tool for an evaluation of the cellular basis of immunity to an infectious disease.

An in vitro assay for T cell immunity to malaria in mice.

After infection with a nonlethal strain of murine malaria (17XNL Plasmodium berghei yoelli), BALB/c mice are then resistant to a lethal strain (17XL P.b. yoelli). BALB/c mice were infected with 17XNL, anc challenged 3 weeks later, after clearing their parasitemias, with 17XL. Three weeks thereafter, spleen cells from such immune animals were used to define an early peaking T-dependent (anti-theta sensitive) antigen-specific proliferative response when incubated in vitro with 17XL infected RBC, or a saline soluble 17XL antigen preparation. T dependent responsiveness of spleen cells from uninoculated control animals to the 17XL antigen preparation was also observed, but demonstrated a much different (delayed) kinetics from that observed with immune cells.

Inbred mice infected with Plasmodium yoelii differ in their antimalarial immunoglobulin isotype response.

Antibodies are known to be important in mediating malarial immunity, but the influence of the various immunoglobulin isotypes on parasite elimination is unclear. The purpose of this study was to provide basic information on the induction of isotype expression in genetically different mice during primary malaria. Parasitaemias and the serum antimalarial IgM, IgG1, IgG2, IgG3 and IgA antibody titres measured in a radioimmunoassay were followed in outbred and 11 inbred strains of mice infected with 17XNL Plasmodium yoelii. Severity of infection, as judged by length of infection, peak parasitaemias and death, was found to differ between the strains. All strains developed rapid IgM responses, but only 3/11 inbred strains produced significant antimalarial IgG1 levels during primary infection. All strains produced an IgG2 response, which developed slightly more quickly in strains with the least severe courses of malaria. A large variation in the IgG3 response was noted between strains. In general, IgG3 antibodies were the first IgG-isotype to appear in serum. They were detected as early as day 8 in strains that developed mild infections but were not present until around day 20 in strains with the most severe cases of malaria. Only one strain produced detectable antimalarial IgA antibodies. These results show that different patterns of isotype expression are induced in inbred strains of mice during primary P. yoelii infection.

Immunoglobulin isotype distribution of malaria-specific antibodies produced during infection with Plasmodium chabaudi adami and Plasmodium yoelii.

The antibody response of mice to Plasmodium chabaudi adami and Plasmodium yoelii has been compared using a solid phase isotype-specific radioimmunoassay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Serological cross-reactivity between these parasites was substantial. Studies using a radioimmunoassay detecting all classes of malaria-specific antibody demonstrated that during the early part of infection it was not possible to distinguish between homologous and heterologous reactions. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 50% or more of the protein antigens detected were apparently shared by both parasites although the intensity of bands was always greater with homologous reactions. However, the distribution of isotypes in the antibody (Ab) response differed in the two infections. P. chabaudi infections were characterized by a predominant and persistent IgM response, moderate IgG2 and IgG3 and little significant IgG1 response during a primary infection. By contrast, IgM antibodies were transient in P. yoelii infection, IgG2 was the predominant isotype, and both IgG1 and IgG3 antibodies were present during a primary infection. These differences in isotypes were also detected when sera were tested on the heterologous antigen extracts suggesting that antigens shared by P. chabaudi and P. yoelii do not necessarily induce similar antibody responses in the two infections.

Protection and recovery in influenza virus-infected mice immunosuppressed with anti-IgM.

BALB/c mice, immunosuppressed from birth with goat anti-mouse IgM, were able to recover from influenza virus infection in the absence of detectable serum and nasal antibody. Recovery was delayed a few days when compared with control animals. Antibody-deficient mice, that had recovered from an initial influenza virus infection, i.e., convalescent mice, were subsequently rechallenged with homologous influenza virus in order to study the importance of nasal and serum antibody in prevention of infection. Convalescent mice were susceptible to reinfection when nasal and serum antibody were not detectable. The mice were resistant to reinfection when serum and/or nasal antibody was detectable by radioimmunoassay. Normal mice that were passively immunized with high titer mouse anti-influenza virus serum were susceptible to challenge with homologous influenza virus. The serum antibody levels in these mice were higher than most of those found in the immune convalescent mice suppressed with anti-IgM, thereby suggesting that the serum antibody, found in convalescent suppressed mice, is not protective. We conclude that 1) mice can recover from influenza virus infection in the absence of detectable levels of nasal and serum antibody, thus indirectly confirming the role of cell-mediated immunity in recovery; 2) serum IgM, IgG2A, IgG2B, IgG3, and probably IgG1 antibody levels are not responsible for protection against influenza virus infection of the upper respiratory tract; and 3) nasal IgA antibody correlates best with protection against reinfection of the upper respiratory tract, but some other locally protective agent cannot be excluded.