Francisco Diaz-Mitoma

The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages.

Interleukin-10 (IL-10), a pleiotropic cytokine that inhibits inflammatory and cell-mediated immune responses, is produced by a wide variety of cell types including T and B cells and monocytes/macrophages. Regulation of pro- and anti-inflammatory cytokines has been suggested to involve distinct signaling pathways. In this study, we investigated the regulation of the human IL-10 (hIL-10) promoter in the human monocytic cell line THP-1 following activation with lipopolysaccharide (LPS). Analysis of hIL-10 promoter sequences revealed that DNA sequences located between base pairs −652 and −571 are necessary for IL-10 transcription. A computer analysis of the promoter sequence between base pairs −652 and −571 revealed the existence of consensus sequences for Sp1, PEA1, YY1, and Epstein-Barr virus-specific nuclear antigen-2 (EBNA-2)-like transcription factors. THP-1 cells transfected with a plasmid containing mutant Sp1 abrogated the promoter activity, whereas plasmids containing the sequences for PEA1, YY1, and EBNA-2-like transcription factors did not influence hIL-10 promoter activity. To understand the events upstream of Sp1 activation, we investigated the role of p38 and extracellular signal-regulated kinase mitogen-activated protein kinases by using their specific inhibitors. SB202190 and SB203580, the p38-specific inhibitors, inhibited LPS-induced IL-10 production. In contrast, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinases, failed to modulate IL-10 production. Furthermore, SB203580 inhibited LPS-induced activation of Sp1, as well as the promoter activity in cells transfected with a plasmid containing the Sp1 consensus sequence. These results suggest that p38 mitogen-activated protein kinase regulates LPS-induced activation of Sp1, which in turn regulates transcription of the hIL-10 gene.

Safe use of the CXCR4 inhibitor ALX40-4C in humans

ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.

Enhancing Oral Vaccine Potency by Targeting Intestinal M Cells

The immune system in the gastrointestinal tract plays a crucial role in the control of infection, as it constitutes the first line of defense against mucosal pathogens. The attractive features of oral immunization have led to the exploration of a variety of oral delivery systems. However, none of these oral delivery systems have been applied to existing commercial vaccines. To overcome this, a new generation of oral vaccine delivery systems that target antigens to gut-associated lymphoid tissue is required. One promising approach is to exploit the potential of microfold (M) cells by mimicking the entry of pathogens into these cells. Targeting specific receptors on the apical surface of M cells might enhance the entry of antigens, initiating the immune response and consequently leading to protection against mucosal pathogens. In this article, we briefly review the challenges associated with current oral vaccine delivery systems and discuss strategies that might potentially target mouse and human intestinal M cells.

Single-Day, Patient-Initiated Famciclovir Therapy for Recurrent Genital Herpes: A Randomized, Double-Blind, Placebo-Controlled Trial

BACKGROUND Orally administered antiviral therapy for genital herpes improves the time to lesion healing and resolves symptoms during an outbreak. Although traditional therapy for a recurrent episode for healthy adults has consisted of twice-daily dosing for 5 days, recent studies have indicated that shorter courses of antiviral therapy are effective. This study was conducted to assess the efficacy and safety of a patient-initiated, single-day regimen of famciclovir therapy, compared with placebo, in immunocompetent adult patients with recurrent genital herpes. METHODS This multicenter, multinational, randomized, double-blind, parallel-group, placebo-controlled study compared single-day, patient-initiated oral famciclovir (1000 mg given twice daily) with placebo for the treatment of recurrent genital herpes. Patients were instructed to initiate therapy within 6 h after onset of prodromal symptoms or genital herpes lesions. RESULTS Famciclovir reduced (P < .001) the time to healing of nonaborted lesions (i.e., those that progressed [corrected] beyond the papule stage) (median time, 4.3 vs. 6.1 days) and all nonaborted and aborted lesions (median time, 3.5 vs. 5.0 days), compared with placebo. The proportion of patients with aborted lesions was larger in the famciclovir group than in the placebo group (23.3% vs. 12.7%; P = .003). Adverse events in the famciclovir group were infrequent overall; most were of mild-to-moderate severity and were similar to adverse events in the placebo group. CONCLUSIONS A single-day regimen of patient-initiated famciclovir treatment was well tolerated and safe, and the healing of recurrent genital herpes lesions occurred approximately 2 days faster than with placebo. Moreover, single-day famciclovir treatment stopped the development or progression of lesions beyond the papule stage. This convenient single-day regimen has the potential for improving patient compliance and satisfaction with therapy.

Total and functional antibody response to a quadrivalent meningococcal polysaccharide vaccine among children

OBJECTIVE To determine total and functional serogroup C antibody response after vaccination with a quadrivalent meningococcal polysaccharide vaccine. DESIGN Prospective, before and after intervention study. SUBJECTS Study subjects were between the ages of 0.5 and 19.9 years, and were eligible for a community-wide public health immunization campaign against Neisseria meningitidis serogroup C. METHODS Total and functional antibody response was measured by enzyme-linked immunosorbent assay and bactericidal assay, respectively. RESULTS One month after vaccination, total capsular polysaccharide antibody significantly increased in all age groups; a significant rise in bactericidal antibody, that correlated with total capsular polysaccharide antibody, was seen in children 18 months of age and older. At 1 year bactericidal antibody titers were maintained but capsular polysaccharide antibody declined substantially in children younger than 5 years. CONCLUSION Total capsular polysaccharide antibody concentration appears to be a useful surrogate measure of bactericidal antibody in children 18 months and older. Children who originally received the vaccine at less than 18 months of age should be considered for revaccination if there is a new or continuing risk of disease. Because of the differences in the total and bactericidal antibodies formed, vaccine efficacy trials are required to define which serologic measures are associated with protection.

Differential Regulation of CD44 Expression by Lipopolysaccharide (LPS) and TNF-α in Human Monocytic Cells: Distinct Involvement of c-Jun N-Terminal Kinase in LPS-Induced CD44 Expression

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-α enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-α-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-α and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-α-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.

Comparative efficacy of famciclovir and valacyclovir for suppression of recurrent genital herpes and viral shedding.

Background: Daily antiviral therapy with famciclovir and valacyclovir has been shown to be effective in reducing both symptomatic and asymptomatic reactivation of herpes simplex virus type 2 (HSV-2) when compared to placebo. However, few comparative studies between the 2 antivirals have been performed. Objectives: To compare the clinical and virologic effects of famciclovir and valacyclovir administered as daily suppressive therapy for persons with genital herpes. Study Design: Two randomized, double-blind, placebo-controlled studies comparing daily famciclovir 250 mg bid with valacyclovir 500 mg qd were performed. Study 1 randomized 320 participants and compared the clinical effect of the drugs given for 16 weeks, and study 2 enrolled 70 HSV-2 seropositive subjects and compared the virologic effect of the drugs given for 10 weeks. Results: In study 1, the time to first recurrence was similar in famciclovir and valacyclovir recipients, hazard ratio (HR) 1.17 (95% CI, 0.78–1.76), but time to first virologically confirmed recurrence was shorter among famciclovir recipients, HR = 2.15 (95% CI, 1.00–4.60). In study 2, HSV was detected on 3.2% of days among famciclovir recipients and 1.3% of days among valacyclovir recipients, relative risk 2.33 (95% CI, 1.18–4.89). Conclusion: Valacyclovir appear to be somewhat better than famciclovir for suppression of genital herpes and associated shedding. Further comparative trials of antiviral drugs for various indications should be performed as acyclovir and penciclovir appear to have different ability to abrogate HSV reactivation.

Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type I (Th1) (interferon‐gamma (IFN‐γ)) and Th2 (IL‐4, IL‐10) type cytokines in peripheral blood lymphocytes (PBL) from HIV patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR) analysis revealed that IFN‐γ mRNA in unstimulated PBL was significantly decreased and IL‐10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n= 30) as compared to patients with > 400 CD4+ T cells/mm3 (n= 6) and normal controls (n= 16). In addition. IL‐10 mRNA levels were inversely associated with IFN‐γ expression. Similar results were obtained by measuring IL‐10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL‐4 and IFN‐7 produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV individuals based on IL‐10 production. PBL from one set of individuals produced low levels of IL‐10 (low IL‐10 producers) whereas the other group produced IL‐10 comparable lo that of normal controls (IL‐10 producers). Production of IL‐4 was significantly reduced in HIV+ individuals with<400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN‐γ by mitogen‐stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulaied and mitogen‐stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.

Interleukin-6 expression in cord blood of patients with clinical chorioamnionitis

The objective of this study was to define whether IL-6 is an early marker of infection in the newborn. To correlate the occurrence of clinical chorioamnionitis with the levels of IL-6 expression in neonates, IL-6 was measured in cord plasma by ELISA and in mononuclear cells by reverse transcriptase-PCR before and after mitogenic stimulation. Eight neonates were included in each of the following four groups: elective cesarean section, uncomplicated normal spontaneous vaginal delivery, delivery after prolonged rupture of amniotic membranes with no evidence of chorioamnionitis, and delivery with evidence of chorioamnionitis. All 32 neonates were clinically well after delivery, and all 16 babies with prolonged rupture of membranes or clinical chorioamnionitis had negative blood cultures. Elevated IL-6 levels were found only in neonates born to mothers with chorioamnionitis (119.7± 33.5 pg/mL versus 2.71 ± 0.59 pg/mL, p< 0.005). Mononuclear cells from five of these neonates expressed no IL-6 mRNA in vivo despite elevated levels of IL-6 in their cord plasma. Cord blood mononuclear cells from healthy term babies were capable of synthesizing IL-6 in vitro in response to stimulation with bacterial lipopolysaccharide. These results suggest that IL-6 levels in cord plasma increased with clinical chorioamnionitis, despite the lack of evidence of infection in the neonates. Therefore, we conclude that, although a high level of IL-6 may be a good marker of chorioamnionitis, it may not be a specific marker of infection in the newborn.

Innovative bioinformatic approaches for developing peptide-based vaccines against hypervariable viruses.

The application of the fields of pharmacogenomics and pharmacogenetics to vaccine design has been recently labeled ‘vaccinomics’. This newly named area of vaccine research, heavily intertwined with bioinformatics, seems to be leading the charge in developing novel vaccines for currently unmet medical needs against hypervariable viruses such as human immunodeficiency virus (HIV), hepatitis C and emerging avian and swine influenza. Some of the more recent bioinformatic approaches in the area of vaccine research include the use of epitope determination and prediction algorithms for exploring the use of peptide epitopes as vaccine immunogens. This paper briefly discusses and explores some current uses of bioinformatics in vaccine design toward the pursuit of peptide vaccines for hypervariable viruses. The various informatics and vaccine design strategies attempted by other groups toward hypervariable viruses will also be briefly examined, along with the strategy used by our group in the design and synthesis of peptide immunogens for candidate HIV and influenza vaccines.