α-Galactosylceramide stimulates splenic lymphocyte proliferation in vitro and increases antibody production in vivo in late neonatal-age mice.

The neonatal stage is characterized by weak responses to various infections and vaccines, thus the development of efficient formulas to improve vaccine effectiveness is of high priority. The glycolipid alpha galactosylceramide (αGalCer) is known as a potent immune modulator due mainly to natural killer (NK) T cell activation. Using a mouse tetanus toxoid (TT) immunization model, we observed that neonatal mice given αGalCer at the time of primary immunization on postnatal day (pnd) 17 had a significantly higher TT-specific immunoglobulin (Ig)M response as well as a memory IgG response, while αGalCer given on pnd 7 resulted in only marginal boosting. Consistently, immunostaining of the spleen sections from αGalCer-treated pnd 17 immunized neonates showed a higher number of Ki67(+) cells in the splenic germinal centre area, suggesting a stronger response after immunization. In-vitro kinetic studies revealed that spleen cells from newborn to pnd 7 neonates did not respond to αGalCer stimulation, whereas cell proliferation was increased markedly by αGalCer after pnd 7, and became dramatic around neonatal pnd 17-18, which was accompanied by increased B, T and NK T cell populations in the spleen. In addition, in pnd 17 spleen cells, αGalCer significantly stimulated the production of NK T cytokines, interleukin (IL)-4 and interferon (IFN)-γ, and promoted the proliferation of CD23(+) B cells, a subset of B cells enriched in germinal centres. These data suggest that αGalCer is an effective immune stimulus in the late neonatal stage, and thus may be useful in translational studies to test as a potential adjuvant to achieve a more efficient response to immunization.

Retinoic acid and tumor necrosis factor-α induced monocytic cell gene expression is regulated in part by induction of transcription factor MafB.

All-trans-retinoic acid (RA), the major active metabolite of vitamin A, is a regulator of gene expression with many roles in cell differentiation. In the present study, we investigated RA in the regulation of MafB, a basic leucine-zipper transcription factor with broad roles in embryonic development, hematopoiesis and monocyte-macrophage differentiation. In RA-treated THP-1 human monocytic cells, MafB mRNA and protein levels were up-regulated by RA dose and time-dependently, while, additionally, RA and tumor necrosis factor (TNF)α, also known to induce monocyte to macrophage differentiation, increased MafB expression synergistically. Screening of potential targets containing Maf recognition elements (MARE motifs) in their promoter regions identified SPOCK1, Blimp1 and CCL2 as potential targets; these genes are related to cell communication, recruitment and differentiation, respectively. Across cell treatments, SPOCK1, Blimp1 and CCL2 mRNA levels were highly correlated (P<0.001) with MafB. ChIP assays demonstrated increased MafB protein binding to MARE elements in the promoter regions of SPOCK1, Blimp1 and CCL2 in RA and TNFα-treated cells, as well as acetylation of histone-H4 in MARE-containing regions, indicative of chromatin activation. Conversely, reducing MafB protein by microRNA silencing significantly decreased the expression of SPOCK1, Blimp1 and CCL2 (P<0.01). Moreover, the reduction in MafB expression and these downstream targets correlated with decreased cell differentiation as determined by cell-surface CD11b expression and phagocytic activity. We conclude that MafB may be a key factor in mediating the ability of RA and TNFα to regulate monocytic cell communication, recruitment and differentiation through regulation of MafB target genes including SPOCK1, CCL2 and Blimp1.

Effects of in utero antiretroviral exposure on mitochondrial DNA levels, mitochondrial function and oxidative stress.

OBJECTIVES: HIV and antiretroviral (ART) exposure in utero may have deleterious effects on the infant, but uncertainty still exists. The objective of this study was to evaluate aspects of mitochondrial DNA (mtDNA) content, mitochondrial function and oxidative stress simultaneously in placenta, umbilical cord blood and infant blood in HIV/ART-exposed infants compared with uninfected controls. METHODS: HIV-1-infected pregnant women and HIV-1-uninfected healthy pregnant controls were enrolled in the study prospectively. Placenta and umbilical cord blood were obtained at delivery and infant blood was obtained within 48 h of delivery. mtDNA content was determined for each specimen. Nuclear [subunit IV of cytochrome c-oxidase (COX IV)]- and mitochondrial (COX II)-encoded polypeptides of the oxidative phosphorylation enzyme cytochrome c-oxidase were quantified in cord and infant blood. Placental mitochondria malondialdehyde (MDA) concentrations were measured as a marker of oxidative stress. RESULTS: Twenty HIV-positive/HIV-exposed and 26 control mother-infant pairs were enrolled in the study. All HIV-infected women and their infants received ART. Placental MDA concentration and mtDNA content in placenta and cord blood were similar between groups. The cord blood COX II:IV ratio was lower in the HIV-positive group than in the controls, whereas the infant peripheral blood mtDNA content was higher in the HIV-exposed infants, but the infant peripheral blood COX II:IV ratio was similar. No infant had clinical evidence of mitochondrial disease or acquired HIV infection. In multivariable regression analyses, the significant findings in cord and infant blood were both most associated with HIV/ART exposure. CONCLUSIONS: HIV-exposed infants showed reduced umbilical cord blood mitochondrial enzyme expression with increased infant peripheral blood mitochondrial DNA levels, the latter possibly reflecting a compensatory mechanism to overcome HIV/ART-associated mitochondrial toxicity.

A high-legume low-glycemic index diet reduces fasting plasma leptin in middle-aged insulin-resistant and -sensitive men.

Fasting leptin and ghrelin levels were measured in 36 insulin-sensitive (IS) and 28 insulin-resistant (IR) men who consumed a legume-enriched low-glycemic index (LG) diet or healthy American (HA) diet in a randomly ordered cross-over feeding study consisting of two 4-week periods. Weight remained stable over the entire study. Fasting plasma leptin was significantly reduced from pre-study levels by both the LG (18.8%, P < 0.001) and HA (16.1%, P < 0.001) diets, whereas fasting ghrelin did not change. By subgroup analysis according to prestudy insulin status, leptin was reduced in IR subjects after both the LG (17.1%, P < 0.01) and the HA (33.3%, P < 0.001) diets, whereas IS subjects responded only after the LG diet (23.1%, P < 0.01). Thus, a legume-rich LG index diet may be a beneficial strategy for reducing circulating leptin concentrations, even under conditions of weight maintenance.

All-trans-retinoic acid and polyriboinosinic : polyribocytidylic acid in combination potentiate specific antibody production and cell-mediated immunity.

Retinoic acid (RA), an active metabolite of vitamin A, may synergize with interferons (IFN) to evoke a heightened immune response, suggesting combination therapy as a promising treatment for various cancers. Recently, we demonstrated a strong synergism between RA and polyriboinosinic : polyribocytidylic acid (PIC), an inducer of IFN, on antibody production in immunocompromised vitamin A-deficient animals. In the present study, we examined whether this combination could potentiate T-cell-dependent antibody production in non-immunocompromised rats. Forty male Lewis rats were treated with 100 microg all-trans-RA, 20 microg PIC, or the combination in either an 11-d study to evaluate antibody production, changes in lymphocyte populations, and cell proliferation, or a 21-hr study to evaluate early changes in lymphocyte populations and gene expression. The combination of RA + PIC significantly potentiated anti-tetanus IgG levels (P < 0.002). Similarly, this combination also increased the numbers of B cells and major histocompatibility complex (MHC) class II+ cells in spleen and lymph nodes, and natural killer (NK) cells in spleen and blood (P < 0.05). RA + PIC-treated rats had significantly higher levels of interleukin (IL)-10, IL-12, and signal transducer and activator of transcription-1 (STAT-1) mRNA (P < 0.05), and STAT-1 protein (P < 0.02). Treatments administered in vivo significantly modulated T-cell proliferation to anti-CD3/phorbol myristyl acetate + IFN-alpha ex vivo. These changes in antibody production, cell distribution, cytokine gene expression, and T-cell proliferation suggest that the combination of RA + PIC stimulates humoral and cell-mediated immunity, and deserves further testing in models of cancer chemoprevention in vivo.

Potentiation of interferon-gamma-stimulated nitric oxide production by retinoic acid in RAW 264.7 cells.

Nitric oxide (NO) production is essential for normal immunity. We have examined the capacity of retinoic acid (RA), a pleiotropic hormone necessary for normal immunity, to modulate NO production in RAW 264.7 cells. NO production induced by suboptimal concentrations of interferon-gamma (IFN-gamma) was significantly greater in cells cultured in low-retinoid medium and treated with all-trans-RA (10(-10) - 10(-6) M, P <0.05), as well as with 9-cis-RA and several retinoids selective for the RA receptor subfamily of nuclear retinoid receptors. Similar results were obtained with lipopolysaccharide and monophosphoryl lipid A as stimuli. The RA-potentiated production of NO was positively correlated with inducible NO synthase (iNOS) protein (r =0.94, P <0.002), although the expression of iNOS mRNA was not altered. We hypothesize that modulation of the macrophage response to suboptimal immune stimuli by physiological concentrations of RA, as observed in these studies, may be important in establishing an optimal balance between T helper (Th) 1- and Th2-mediated immunity.

Fatty acid delta(5)-desaturase mRNA is regulated by dietary vitamin A and exogenous retinoic acid in liver of adult rats.

Delta(5)-Desaturase (D5D) catalyzes the Delta(5,6) desaturation of dietary essential fatty acids of the n-6 and n-3 series. By subtraction hybridization of vitamin A (VA)-deficient and control rat liver cDNA libraries, we isolated a 106-bp cDNA fragment that proved to be homologous to human liver D5D cDNA and used it as a probe to analyze rat D5D mRNA and clone the rat full-length cDNA. Delta(5)-Desaturase mRNA was threefold more abundant in liver from VA-deficient rats than in liver from VA-sufficient rats and was expressed dose dependently when dietary VA was varied (VA marginal > control > VA supplemented). Treatment of VA-deficient rats with all-trans-retinoic acid lowered the level of expression of D5D mRNA toward that of VA-sufficient rats. The 3413-bp full-length D5D cDNA cloned from rat liver contains an open reading frame of 447 amino acid residues sharing 92% similarity with its human counterpart. Expression of this cDNA in HEK293T cells incubated with dihomo-gamma-linolenic acid (20:3, n-6) resulted in a significantly increased ratio of the product, arachidonic acid (20:4, n-6), to substrate in cell lipid extracts. Delta(5)-Desaturase mRNA is expressed in relatively high abundance in rat adrenal gland and mammary tissue and moderately in liver, kidney, lung, spleen, thymus, brain, and eye. The regulation of D5D by VA could be important for growth and development, and reproduction, as well as in the control of inflammation.

Subjectively perceived quality of life after coronary artery bypass surgery.

BACKGROUND: Judgment of quality of life after coronary artery bypass surgery is usually based on objective measures of cardiovascular status. Quality of life cannot be determined solely objectively because such indicators do not explain how persons perceive and experience their lives. OBJECTIVES: To assess the quality of life and mood state over time in patients undergoing coronary artery bypass grafting and to improve understanding of subjective perceptions of well-being and how these perceptions change over time. METHODS: Three questionnaires, the Quality of Life Index, the Medical Outcomes Study 36-Item Short-Form Health Survey, and the Profile of Mood States, were administered at 3 different times (before surgery, 6 weeks after surgery, and 3 months after surgery) to a convenience sample of hospitalized adults undergoing coronary artery bypass surgery for treatment of coronary artery disease. RESULTS: For all 3 questionnaires, responses differed significantly over time. Mean scores were significantly different over time for total mood disturbance (P = .03), the socioeconomic domain of the Quality of Life Index (P = .02), and the physical functioning (P = .004), vitality (P = .007), and social functioning (P = .002) dimensions of the 36-item short-form survey. CONCLUSIONS: Subjective perceptions of physical and psychological well-being changed significantly from before surgery to 3 months after surgery. Measures of mood state, physical functioning, vitality, and social functioning improved significantly over time. However, satisfaction with the socioeconomic domain decreased significantly from before surgery to 3 months after surgery.

Lecithin:retinol acyltransferase from mouse and rat liver. CDNA cloning and liver-specific regulation by dietary vitamin a and retinoic acid.

Lecithin:retinol acyltransferase (LRAT), present in microsomes, catalyzes the transfer of the sn-1 fatty acid of phosphatidylcholine to retinol bound to a cellular retinol-binding protein. In the present study we have cloned mouse and rat liver LRAT cDNA and tested the hypothesis that LRAT mRNA, like LRAT activity, is regulated physiologically in a liver-specific manner. The nucleotide sequences of mouse and rat liver LRAT cDNA each encode a 231-amino acid protein with 94% similarity between these species, and approximately 80% similarity to a cDNA for LRAT from human retinal pigment epithelium. Expression of rat LRAT cDNA in HEK293T cells resulted in functional retinol esterification and storage. RNA from several rat tissues hybridized with liver LRAT cDNA. However, LRAT mRNA was virtually absent from the liver of vitamin A-deficient animals, while being unaffected in intestine and testis. LRAT mRNA was rapidly induced by retinoic acid (RA) in liver of vitamin A-deficient mice and rats (P < 0.01). LRAT mRNA and enzymatic activity were well correlated in the same livers of rats treated with exogenous RA (r = 0.895, P < 0.0001), and in a dietary study that encompassed a broad range of vitamin A exposure (r = 0.799, P < 0.0001). Liver total retinol of <100 nmol/g was associated with low LRAT expression (<33% of control). We propose that RA, derived exogenously or from metabolism, serves as an important signal of vitamin A status. The constitutive expression of liver LRAT during retinoid sufficiency would serve to divert retinol into storage pools, while the curtailment of LRAT expression in retinoid deficiency would maintain retinol for secretion and delivery to peripheral tissues.

Regulation of CYP26 (cytochrome P450RAI) mRNA expression and retinoic acid metabolism by retinoids and dietary vitamin A in liver of mice and rats.

Retinoic acid (RA), through nuclear retinoid receptors, regulates the expression of numerous genes. However, little is known of the biochemical mechanisms that regulate RA concentration in vivo. CYP26 (P450RAI), a novel cytochrome P450, is expressed during embryonic development, induced by all-trans RA, and capable of catalyzing the oxidation of [3H]RA to polar retinoids including 4-oxo-RA. Here we report that CYP26 expression in adult liver is regulated by all-trans RA and dietary vitamin A, and is correlated with the metabolism of all-trans RA to polar metabolites. In normal mouse and rat liver, CYP26 mRNA was barely detectable; however, after acute treatment with all-trans RA CYP26 mRNA and RA metabolism by liver microsomes were significantly induced. Aqueous-soluble RA metabolites were detected, but their formation was not induced. The expression of retinoid receptors, RAR-gamma and RXR-alpha, was not changed after RA treatment in vivo. In a model of chronic vitamin A ingestion during aging, CYP26 mRNA expression, determined by Northern blot and RT-PCR analysis, increased progressively with dietary vitamin A (P<0.0001; marginal < control < supplemented) and age (P<0.003). The relative expression of CYP26 mRNA was positively correlated with liver total retinol (log10), ranging from undetectable CYP26 expression at liver retinol concentrations below approximately 20 nmol/g to a three- to fourfold elevation at concentrations >10,000 nmol/g (r=0.90, P<0.0001). We conclude that CYP26 expression and RA metabolism are regulated in adult liver not only acutely by RA administration, as may be relevant to retinoid therapy, but under chronic dietary conditions relevant to vitamin A nutrition in humans.

Chronopharmaceutical drug delivery from a pulsatile capsule device based on programmable erosion.

We report the development of a chronopharmaceutical capsule drug delivery system capable of releasing drug after pre-determined time delays. The drug formulation is sealed inside the insoluble capsule body by an erodible tablet (ET). The release time is determined by ET erosion rate and increases as the content of an insoluble excipient (dibasic calcium phosphate) and of gel-forming excipient (hydroxypropylmethylcellulose; HPMC) increases. The time-delayed release of a model drug (propranolol HCI) was investigated by dissolution testing (USP XXIII paddle method). Both composition and weight of ET influence the time of drug release. Moreover it was found that drug release was controlled by the quantity of HPMC, irrespective of lactose content within the tablet weight range 80-160 mg, when above a threshold concentration of 20% HPMC. Programmable pulsatile release has been achieved from a capsule device over a 2-12-h period, consistent with the demands of chronotherapeutic drug delivery. The time of drug release can be controlled by manipulation of tablet formulation.

An analysis of breast cancer surgery after free transverse rectus abdominis myocutaneous (TRAM) flap reconstruction.

BACKGROUND: Breast reconstruction is currently offered on a more routine basis to patients after mastectomy for breast cancer. This paper analyzes the outcomes of breast cancer surgery, and the results and effects of breast reconstruction using free TRAM flaps. METHODS: A retrospective review of 75 consecutive patients who had free transverse rectus abdominis myocutaneous (TRAM) flap breast reconstruction after breast cancer surgery was performed. A total of 92 free TRAM flaps were performed on 75 patients in Victoria, British Columbia, from January 1992 to May 1999. Thirty-three patients (44%) underwent primary breast cancer surgery and an immediate reconstruction (7 bilateral and 27 unilateral) and 42 patients (56%) had delayed reconstruction (10 bilateral and 32 unilateral). RESULTS: Twenty- one patients (28%) had stage 0 disease, 20 (26.7%) had stage I disease, 17 (22.7%) had stage IIA disease, 12 (15%) had stage IIB disease, and 4 (5.3%) had stage IIIA disease. In 1 patient the stage of disease was unknown. The mean patient age was 49.4 years (range 33 to 73). Of the patients undergoing immediate reconstruction 3 had postoperative chemotherapy and 1 had postoperative radiotherapy. Three patients had combined chemoradiotherapy. In none of these cases was the adjuvant therapy delayed by the reconstructive surgery. Overall mean follow-up time from cancer diagnosis was 56.8 months and from the time of TRAM flap reconstruction, 36.7 months. To date, 5 recurrences have been detected (6.6%). Mean time between reconstruction and detection of recurrence was 22.8 months. Detection of recurrence was achieved clinically and was not impaired in any of the cases by the presence of the free flap. Patient satisfaction was assessed via a telephone survey, with 93% of patients pleased with the cosmetic results of their surgery. CONCLUSIONS: For those patients with breast cancer requiring mastectomy, free TRAM flap reconstruction is a safe, cosmetically acceptable surgical alternative that impairs neither effective breast cancer surgery nor detection of recurrent disease.

Retinoic acid and polyriboinosinic acid act synergistically to enhance the antibody response to tetanus toxoid during vitamin A deficiency: possible involvement of interleukin-2 receptor-beta, signal transducer and activator of transcription-1, and interferon regulatory factor-1.

Antibody responses to T cell-dependent antigens are reduced during vitamin A (VA) deficiency and restored by retinoids. To test whether retinoic acid (RA) and polyinosinic:polycytidylic acid (PIC), an inducer of interferons, can increase specific antibody production, VA-deficient rats were treated with all-trans-RA, PIC, or both at the time of primary immunization with tetanus toxoid. VA-deficient rats produced low primary and secondary anti-tetanus IgG responses (P<.001 vs. VA-sufficient controls). Both responses were increased synergistically by RA plus PIC (P<.0001). In VA-deficient spleens, mRNAs were low for interleukin (IL)-2 receptor-beta, interferon regulatory factor-1, and signal transducer and activator of transcription 1. Each, however, was induced by RA plus PIC (P<.0001 vs. controls). Conversely, IL-12 and IL-10 mRNAs were elevated in VA deficiency and were induced by PIC and suppressed by RA. Thus, RA plus PIC appears to be a promising combination for stimulating antigen-specific immunity. Several molecular factors identified here may partially account for the observed enhancement.

Iron status and stores decline with age in Lewis rats.

In the context of a larger study examining the interaction of vitamin A (VA) status and age on immune function, we examined age-related changes in hematologic and iron status variables in male Lewis rats. Animals were fed a nutritionally adequate purified diet containing either 0.35 (marginal), 4.0 (control) or 50 (supplemented) mg retinol equivalents (as retinyl palmitate) per kg of diet from the time of weaning until killing at 8-10 (middle-aged) or 20-22 (old) mo of age. Neither VA nor VA and age interaction effects were significant for most iron variables examined. After controlling for body weight, old rats had significantly lower hemoglobin, hematocrit and plasma iron than middle-aged rats. This decrease in hematologic and transport iron variables was not accompanied by a shift of iron into other storage compartments. Old rats also had significantly lower total iron content and iron concentration in liver, spleen and bone marrow. Hemosiderin iron in marrow smears correlated significantly (r = 0.43-0.76, P: < 0.05) with chemical estimates of iron in storage, transport and functional pools. Old rats also tended to have less stained iron in femur marrow smears. Thus, body iron in functional, transport and storage compartments, namely the liver, spleen and bone marrow, were significantly lower in old than in middle-aged rats. Although iron stores and status are usually considered to increase with advancing age, our data show a consistent pattern of lower hematologic and storage iron variables in old than in middle-aged Lewis rats. Future research is indicated to understand the biology and functional consequences of the observed age-associated decline in body iron.

Relation of serum retinol to acute phase proteins and malarial morbidity in Papua New Guinea children.

BACKGROUND: Acute phase proteins (APPs) are associated with malaria-induced hyporetinemia (serum retinol <0.70 micromol/L); however, the degree of the association is not well documented. OBJECTIVE: The association between malaria-induced hyporetinemia and APPs was assessed. DESIGN: In a cross-sectional study, 90 children with serum retinol concentrations from <0.35 to >1.05 micromol/L were selected from children in a clinical trial of vitamin A supplementation. Serum was collected before treatment allocation. Retinol binding protein (RBP) concentrations were determined by radioimmunoassays, and transthyretin, alpha(1)-acid glycoprotein (AGP), alpha(1)-antichymotrypsin, C-reactive protein (CRP), haptoglobin, and albumin concentrations by radial immunodiffusion assays. RESULTS: Children in the subsample had high rates of splenomegaly and Plasmodium-positive blood-smear slides (P < 0.01); AGP (Pearson's r = -0.40, P < 0.001) and CRP (r = -0.21, P = 0.04) were inversely correlated with retinol. The negative APPs RBP, transthyretin, and albumin were positively and significantly associated with retinol. All APPs, except alpha(1)-antichymotrypsin, were significantly correlated with splenomegaly. Of the positive APPs, AGP correlated with CRP (r = 0.37, P < 0.001), indicating chronic inflammation. In a stepwise regression analysis, 73% of retinol's variability was explained by RBP and transthyretin. The model predicted that a 1-SD increase in RBP or transthyretin increases retinol by approximately 0.38 or 0.47 micromol/L, respectively, whereas an equivalent increase in AGP decreases retinol by 0.12 micromol/L. CONCLUSIONS: The RBP-transthyretin transport complex of retinol is not altered by inflammation. Positive APPs are useful markers of type and severity of inflammation; however, except for AGP, it is unlikely that they can correct for malaria-induced hyporetinemia.

Regulation of hepatic vitamin A storage in a rat model of controlled vitamin A status during aging.

It is currently unknown whether the capacity of the liver to esterify and store vitamin A (VA) changes as a function of long-term VA intake or age. The objective of this study was to investigate whether age and/or VA status are factors for the hepatic expression of cellular retinol-binding protein (CRBP), the esterification of retinol by lecithin:retinol acyltransferase (LRAT) and the accumulation of VA and lipids in liver. Two factors, VA intake and age, were studied in a 3x3 design. Diets denoted as VA-marginal, control and supplemented contained 0.35, 4 and 25 mg retinol equivalents/kg diet, respectively; male Lewis rats were fed these diets from weaning until the ages of 2-3 mo (young), 8-10 mo (middle-aged) and 18-20 mo (old) (n = 6/group. Liver CRBP mRNA differed (two-way ANOVA) with dietary VA (P<0.0001) and age (P<0.05). Hepatic LRAT activity increased with dietary VA (P<0.0001). Age was not a factor (P = 0.47) although there was an interaction of age and dietary VA (P<0.0001). Hepatic LRAT activity was correlated (r = 0.633, P<0.0001) with plasma retinol at physiologic concentrations. In VA-supplemented rats of all ages, the plasma molar ratio of total retinol:retinol-binding protein (RBP) exceeded 1, and liver VA and total lipid concentrations were elevated. However, tests of liver function had previously been shown to be within normal values. Thus, the capacity of the liver for retinol esterification by LRAT was not diminished by age or the accumulation of VA and other lipids. We conclude the following: 1) hepatic LRAT activity is regulated across a broad, physiologic range of dietary VA; 2) LRAT activity is regulated throughout life; and 3) the capacity for hepatic VA storage is high throughout life.

All-trans-retinoic acid and polyriboinosinoic:polyribocytidylic acid cooperate to elevate anti-tetanus immunoglobulin G and immunoglobulin M responses in vitamin A-deficient lewis rats and Balb/c mice.

Vitamin A (VA) deficiency compromises antibody responses to T-cell-dependent antigens such as tetanus toxoid, but this effect can be reversed through administration of retinol or retinoic acid (RA). To test whether RA and polyriboinosinioc : polyribocytidylic acid (PIC), a known inducer of several forms of interferon (IFN), can cooperate to increase specific immunoglobulin (Ig)G and IgM production during VA deficiency, rats and mice were made VA-deficient, immunized with TT and treated with all-trans-RA, PIC or their combination. VA-deficient rats produced low primary and secondary anti-tetanus IgG responses (VA-deficient controls v. VA-sufficient controls P < 0.001), although total IgG was slightly elevated when compared with VA-sufficient control rats. Although RA administered alone elevated antibody production during VA deficiency to control levels, RA combined with PIC synergistically enhanced these responses (RA and PIC group v. all other groups P < 0.0001). In contrast, Balb/c mice maintained on a VA-deficient diet and immunized in a similar fashion showed no impairment in antigen-specific IgG levels, but treatment with a combination of RA and PIC still evoked an additive enhancement in antigen-specific antibody production. Additionally, RA and PIC administration to VA-sufficient mice resulted in elevated antibody responses, suggesting that this combination should be evaluated further for its immuno-stimulatory effects.

Effect of pH on rate of interfacial inactivation of serine proteases in aqueous-organic systems.

We studied the inactivation of trypsin and alpha- and beta-chymotrypsin by passage of droplets of tridecane though their aqueous solutions. The mechanism involves contact with the interface, because the loss of activity is proportional to the total area exposed. The rates of inactivation vary up to fivefold over the pH range 3 to 10. However, there is no clear maximum at the isoelectric point (pI) of each enzyme, where the amount of protein adsorbed is usually found to be highest. This is probably because, at the pI, there is also a minimum in structural alteration on adsorption. There may be a weak correlation with pH effects on foamability of the enzyme solutions, a parameter reported to reflect the "hardness" of different proteins, which controls their interfacial unfolding. The pH dependence of both inactivation and hardness cautions against attempts to correlate inactivation of different enzymes with a single value of a parameter such as adiabatic compressibility. There is no correlation between the effects of pH on interfacial inactivation and those reported in the literature on irreversible inactivation in concentrated urea or at high temperature.