Rotavirus causes gastroenteritis in children. The introduction of vaccines protecting children against some rotavirus serotypes induced a redistribution of circulating serotypes in children. The mechanisms driving this redistribution are not well understood. One hypothesis is that remaining unvaccinated children are more exposed to adults who have a different serotype distribution. The presented model tests the plausibility of this hypothesis. A transmission model was developed with two serotypes circulating in children and adults. Transmission was considered within each population and from adults to children. The model assumed closed populations but with different children and adult serotype distributions prior to vaccination. Vaccination protecting children against both serotypes was introduced, after which the model estimated shifts in the new postvaccine serotype distribution equilibrium. Default parameters were identified, and scenario analyses were tested on vaccine coverage rate, levels of adult-to-child transmission rates, and relative population sizes. The distributions between the two serotypes prior to vaccination were 25%:75% in children and 50%:50% in adults, and the proportions infected were 43% and 33%, respectively. For the default parameters, 80% vaccine coverage reduced the proportion of children infected by 92% and shifted the child serotype distribution at equilibrium toward the adult distribution to 47%:53%. Similar results were observed for all parameter scenarios, with the serotype distribution among infected children shifting closer to the adult distribution as vaccine coverage increased. The model demonstrates that serotype distribution in children after vaccination may be influenced by the adult serotype distribution, with higher vaccine coverage shifting the child distribution closer to the adult distribution. As adult-to-child infection becomes more common than child-to-child infection, the adult serotype distribution becomes more prominent in the child population. These results support the hypothesis that rotavirus exposure from adult populations contributes to observed shifts in rotavirus serotype distributions in children after vaccine introduction.

Background Imiquimod, a potent immunomodulator, not having a direct antiproliferative activity, was found to be effective in genital and cutaneous premalignancies and malignancies. As tumor development depends on blood vessel supply, the inhibition of angiogenesis could be responsible for the antitumor activity.Objective To find in a murine model whether imiquimod has antiangiogenic activity and whether this activity is mediated by locally induced cytokines.Methods The study was performed in two cell lines: Skv human keratinocytes containing multiple integrated copies of HPV16 derived from bowenoid papulosis, and murine L1 lung sarcoma cells of Balb/c mice. The murine model of cutaneous angiogenesis was used to assess and count the new blood vessel formation. The mice were immunosuppressed by a total body X-ray irradiation and treated with 5% or 2.5% imiquimod cream before or after induction of angiogenesis with intradermally injected tumor cell suspension. In some experiments the mice were, in addition, treated intraperitoneally with monoclonal antibodies against murine IFNalpha, TNFgamma or IL-18.Results Topical application of imiquimod on the murine skin resulted in reduction of angiogenesis (P < 0.001) induced by intradermal injection of both human and mouse tumor cells, more pronounced when 5% cream was applied on three consecutive days. Antibodies against murine IFNgamma, TNFalpha and IL-18 completely abolished the inhibitory effect of imiquimod on angiogenesis induced by murine L1 sacroma cells. When human Skv cells were used in angiogenesis assay, the effect of imiquimod was abolished by antibodies against IL-18 but not against TNFalpha, which may be due to overproduction of TNFalpha by Skv cells.Conclusions Antiangiogenic effect of imiquimod was found to be mediated by IL-18, probably through promoting production of INFgamma, the most important inhibitor of angiogenesis.

Background Actinic keratoses (AKs) are premalignant lesions that can progress into squamous cell carcinoma. Imiquimod, which belongs to the new class of immune-response modifiers, was recently shown to be effective in the treatment of AKs. The underlying mechanisms are not fully understood.Objectives To study the expression of individual genes in uninvolved skin and AKs before therapy and to elucidate the way in which the expression of these genes is influenced by imiquimod therapy.Methods We treated 13 patients with AK with imiquimod and compared gene expression before, during (five patients) and after (eight patients) therapy with that in uninvolved skin. We analysed genes coding for inflammatory cytokines or their receptors, adhesion molecules, anti-apoptotic proteins, p53 and toll-like receptors (TLRs) by reverse-transcriptase polymerase chain reaction.Results Comparing uninvolved skin and untreated AK, we found significant differences in the expression of interleukin (IL)-6, hurpin, TLR7 and TLR8. During imiquimod therapy, we detected a further upregulation of interferon-alpha, IL-6, IL-10 receptor 1 and TLR7. In contrast, two anti-apoptotic genes, hurpin and HAX-1, were downregulated. We did not detect significant differences in gene expression for p53, tumour necrosis factor-alpha and alpha- and beta-catenins. Clinically, the upregulated expression of the proinflammatory cytokines correlated with the local inflammation induced by imiquimod.Conclusions Our results indicate that specific differences in gene expression are detectable between AK and uninvolved skin. Imiquimod influenced the expression of most genes analysed in this study. This work extends previous findings on the effects of imiquimod on gene regulation in AKs.

BACKGROUND:Imiquimod is a local immune response modifier that has demonstrated potent antiviral and antitumor activity. It enhances innate and acquired immune responses via endogenous cytokine production and has proven efficacious in clearing superficial basal cell carcinoma (sBCC).OBJECTIVE:To evaluate the mechanisms by which topical imiquimod treatment leads to sBCC clearance in vivo.DESIGN:A pilot, open-label, nonrandomized study.SETTING:Zurich, Switzerland.PATIENTS:Six persons 18 years or older who had nonrecurrent primary tumors that had not undergone previous biopsy or treatment but were suitable for treatment by surgical excision. The tumors were located on the scalp, extremities, or trunk; had a minimum diameter of 1 cm and a maximum diameter of 2 cm; and were clinically and histologically consistent with sBCC.INTERVENTIONS:Daily application of 5% imiquimod cream 5 times per week for a maximum of 6 weeks. When the tumor began to show signs of erosion, it was surgically excised.OUTCOME MEASURES:Parameters reflecting tumor apoptotic status (Bcl-2), expression of death receptors (Fas and Fas ligand [FasL]), intercellular adhesion molecule (ICAM) 1, immunosuppressive microenvironment (interleukin 10), and antigen presentation machinery (transporter associated with antigen presentation [TAP] 1) before and after imiquimod treatment were evaluated. The changes in the interferon gamma messenger RNA (mRNA) levels relative to CD4 and CD8 mRNA were assessed using quantitative polymerase chain reaction.RESULTS:Tumor cells became more susceptible to apoptosis through decreased Bcl-2 expression after treatment with 5% imiquimod cream. Inflammatory infiltrate developed rapidly (within 3 to 5 days after treatment initiation) and was associated with the enhanced expression of ICAM-1. This early response tended to be a mixed cellular response of macrophages and lymphocytes. Interferon gamma was produced by CD4 and CD8 T cells. Imiquimod treatment induced a massive increase in macrophage peritumoral and intratumoral infiltration. Interleukin 10 was produced by infiltrating cells but was not produced by tumor cells. Tumor expression of TAP-1 and Fas/FasL appeared to be unaffected in the first 5 days of treatment.

BACKGROUND:Actinic keratoses (AKs) are precancerous epidermal lesions found most frequently on areas of the skin exposed to the sun. Several case studies published recently have indicated that 5% imiquimod cream, currently licensed for the treatment of genital warts, may be an effective treatment for AK.OBJECTIVE:To assess the efficacy and safety of imiquimod for the treatment of AK.DESIGN:Patients in this randomized, double-blind, vehicle-controlled study applied 5% imiquimod cream or vehicle to AK lesions 3 times per week for a maximum of 12 weeks or until lesions had resolved. In the event of an adverse reaction, application of imiquimod was reduced to 1 or 2 times per week. Rest periods were also allowed if necessary.SETTING:A specialized outpatient dermatology clinic within a state-funded hospital in Germany.PATIENTS:The study population was aged 45 to 85 years. Of 52 patients screened, 36 men and women with AK confirmed by histological diagnosis were enrolled. Patients were excluded from the study if they did not have a histological diagnosis for AK, if they were older than 85 years, or if they did not comply with the protocol. All patients had responded to a notice asking for volunteers.MAIN OUTCOME MEASURES:The number and appearance of lesions were evaluated before, during, and after treatment. All adverse effects were recorded.RESULTS:Lesions treated with 5% imiquimod cream were clinically cleared in 21 (84%) of 25 patients and partially cleared in 2 (8%). Clearance was histologically confirmed 2 weeks after the last application of imiquimod in all patients clinically diagnosed as lesion free. Only 10% of patients treated with imiquimod were clinically diagnosed with recurrence 1 year after treatment. No reduction in the size or number of AK lesions was observed in vehicle-treated patients. Adverse effects reported by patients treated with imiquimod included erythema, edema, induration, vesicles, erosion, ulceration, excoriation, and scabbing. However, imiquimod was well tolerated since all patients completed the 12-week treatment. Only a few, mild adverse reactions to the vehicle cream were reported.CONCLUSION:Application of 5% imiquimod cream for 12 weeks is an effective and well-tolerated treatment for AK.

Background Actinic keratoses (AK) are premalignant lesions, which are routinely treated by destructive procedures such as cryotherapy, electrodessication or topical 5-fluorouracil.Objectives The aim of this study is to report six cases of AK treated with a potential new topical therapy, imiquimod.Methods Subjects included in this study had suffered with recurrent AK for between 5 and 16 years. All six men were treated with imiquimod 5% cream three times a week for 6-8 weeks. In the event of a local skin reaction treatment was modified to two times per week.Results All the AK lesions were successfully cleared after treatment with imiquimod cream 5% for 6-8 weeks. Histologically, no apparent signs of persisting AK could be detected, and no recurrences were reported during follow up.Conclusions This study suggests that imiquimod may be useful as a new therapy for the treatment of actinic keratoses.

Immunomodulators include both immunostimulatory and immunosuppressive agents. Only recently have the basic mechanisms of topical immunotherapy been elucidated. Besides topical contact sensitizers (eg, diphencyprone or dinitrochlorobenzene), newer agents of the imidazoquinoline family such as imiquimod and resiquimod act by inducing cytokine secretion from monocytes or macrophages (interferon-12, interleukin-12, tumour-necrosis factor-α). The locally generated immune milieu leads to a Th1-dominance and cell-mediated immunity that have been used clinically to treat viral infections such as human papillomavirus (HPV), herpes simplex virus (HSV), mollusca, and cancerous lesions including initial squamous cell and basal cell carcinoma in immunocompetent and immunosuppressed patients. While these agents improve antigen-presentation by dendritic cells, they also act on B cells and lead to the synthesis of antibodies such as IgG2a much like the recently discovered immunostimulatory CpGsequences that stimulate innate immunity. These sequences act as “danger signals” since they occur in bacterial and viral DNA, but are selectively methylated and inactivated in the mammalian genome. They share the induction of the same cytokines as imidazoquinolines but they show different magnitudes and kinetics of response. Topical immunotherapy with immunostimulatory agents shows potential for effective and patient-friendly treatment of inflammatory, infectious, and cancerous skin diseases. Immunoenhancers such as imidazoquinolines and CpG-sequences also have adjuvant properties that could improve conventional (protein) and DNA vaccination against cancer, atopy, and allergies.

Activated macrophages exert strong arginase (ASE) activity that converts L-arginine into ornithine, the key precursor for putrescine and polyamine biosynthesis. Macrophages were previously also shown to generate nitric oxide that is derived from the guanido group of arginine by the oxidative deiminase (OAD) reaction. In view of the physiological importance of ornithine and putrescine, we now investigated whether interferon-gamma (IFN-gamma), a principal stimulator of the OAD activity, may lead to the accumulation of the deiminated derivative citrulline at the expense of ornithine production, or whether the carbon backbone could be reutilized for the production of arginine and ornithine. Our experiments show that murine peritoneal macrophages treated with IFN-gamma in combination with tumor necrosis factor (TNF) or bacterial lipopolysaccharide (LPS) generate substantial amounts of citrulline as identified by amino acid analyzer and by thin-layer chromatography. Also, labeled citrulline is generated from [14C]L-arginine but not from [14C]L-ornithine. This suggests that macrophages have little or no capacity to convert ornithine into arginine. In the absence of IFN-gamma, TNF and LPS stimulate the conversion of arginine into ornithine but not citrulline. However, when TNF or LPS stimulated macrophages are simultaneously treated with IFN-gamma, ornithine production is relatively inhibited by the strong OAD reaction that competes with the ASE reaction for its substrate L-arginine. IFN-gamma thus down-regulates the availability of ornithine and putrescine. The lipid A precursor IA also induces, in conjunction with IFN-gamma, the production of citrulline but fails to stimulate the generation of ornithine.

The structural elements of Salmonella typhimurium lipopolysaccharides (LPS) that are able to stimulate peritoneal macrophages to produce increased amounts of prostaglandin E2, ornithine, and citrulline, agents known to modulate immune responses, are described. Two different incomplete lipid A structures which lack the carbohydrate portion, the nonhydroxylated fatty acids lauric acid and myristic acid (lipid A precursor IB), and additional palmitic acid (lipid A precursor IA) stimulated increased prostaglandin E2 synthesis but were unable to augment ornithine and citrulline production at concentrations of up to 0.5 microgram/ml. Acyl-deficient smooth LPS containing lipid A precursors IA and IB substituted by the complete carbohydrate region were able to augment prostaglandin E2 and ornithine production but failed, even at a high concentration (0.5 microgram/ml), to stimulate citrulline production. Moreover, Re glycolipids and smooth intact LPS containing the lipid A region with 3-acyloxyacyl residues possessed all of the structural requirements to induce increased prostaglandin E2, ornithine, and citrulline synthesis. Finally, all of the LPS structures, including lipid A precursors IA and IB stimulated, in combination with gamma interferon, production of citrulline with similar efficiencies. These results demonstrate that LPS contains various substructures including regions of the carbohydrate and lipid A structure that can deliver signals for the activation of peritoneal macrophages. Signals for partial activation of macrophages to produce prostaglandins and ornithine can be delivered by acyl-deficient LPS structures. In contrast, full activation of macrophages to produce citrulline requires an additional signal that is delivered by 3-acyloxyacyl residues of the lipid A region or gamma interferon.

There is an increasing body of evidence that T cell-mediated immune response is regulated by a variety of small molecular weight products of macrophages. One of the best known immunoregulatory products in this context is prostaglandin E2 (PGE2) which is known to regulate both T cell and macrophage functions. Recently, ornithine, cysteine, and lactate have also been recognized as immunoregulatory mediators. In analogy to the hormone-like cytokines and lymphokines, all these substances are produced by immunologically relevant cells (macrophages) at a variable and regulated rate; and they have been shown to regulate the functional activities of other cells (T cells and macrophages). This brief review describes the key observations that underscore the important regulatory role of these metabolites in physiological and pathological conditions.

Macrophages consume cystine and generate approximately equivalent amounts of acid-soluble thiol. Stimulation of macrophages with bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF) strongly augments the amount of thiol released into the culture supernatant. Cysteine constitutes most of the acid-soluble thiol. The intracellular glutathione level and the DNA synthesis activity in mitogenically stimulated lymphocytes are strongly increased by either exogenously added cysteine, or (syngeneic) macrophages. This cysteine dependency is observed even in the presence of relatively high extracellular cystine concentration as they occur in the blood plasma. The extracellular cysteine concentration also has a strong influence on the intracellular glutathione concentration, viability, and DNA synthesis of cycling T cell clones. Moreover, the cysteine concentration in the culture medium on Day 3 and Day 4 of a 5-day allogeneic mixed lymphocyte culture (i.e., in the late phase of incubation) has a strong influence on the generation of cytotoxic T cell activity, indicating that regulatory effects of cysteine are not restricted to the early phase of the blastogenic response. The inhibitory effect of cysteine starvation on the DNA synthesis of the T cell clones and on the activation of cytotoxic T lymphocytes can be explained essentially by the depletion of intracellular glutathione, since similar effects are observed after treatment with buthionine sulfoximine (BSO), a specific inhibitor of the glutathione biosynthesis. BSO has practically no influence, however, on the N alpha-benzyloxycarbonyl Ne-t-butyloxycarbonyl-L-lysine-thiobenzyl-ester (BLT)-esterase activity and hemolytic activity of the cell lysates from cytotoxic T cells against sheep red blood cells (perforin activity). Taken together, our experiments indicate that cysteine has a regulatory role in the immune system analogous to the hormone-like lymphokines and cytokines. It is released by macrophages at a variable and regulated rate and regulates immunologically relevant functions of lymphocytes in the vicinity.

Bacterial lipopolysaccharides (LPS) stimulate the ability of macrophages to convert arginine into ornithine. This effect is inhibited by the cycloxygenase inhibitor indomethacin and reconstituted by application of exogenous prostaglandin E2 (PGE2) or cholera toxin, i.e. two substances which are known to raise the intracellular concentration of cyclic AMP. Moreover, the LPS-mediated effect is augmented in a synergistic manner by PGE2, cholera toxin and the dibutyryl-derivative of cyclic AMP. Lipid A precursor IA, which lacks lauric, myristic and palmitic acids, and lipid A precursor IB, which is devoid of lauric and myristic acids of the complete lipid A structure, are capable of augmenting PGE2 synthesis but do not stimulate the conversion of arginine into ornithine. Taken together, our experiments suggest that the LPS-induced production of PGE2 and the PGE2-induced increase of the intracellular cAMP concentrations are essential elements of an autoregulatory loop that controls the LPS-mediated stimulation of the ornithine production by macrophages. The stimulation of the autoregulatory loop is necessary but not sufficient for this effect, indicating that an additional signal is required which is provided by the complete lipid A structure but not by the incomplete lipid A structures IA and IB. This additional signal can be provided by a lipid A structure containing the 3-acyloxyacyl residues with lauric and myristic acids.