Herpes simplex virus type-2 stimulates HIV-1 replication in cervical tissues: implications for HIV-1 transmission and efficacy of anti-HIV-1 microbicides.

Herpes Simplex virus Type-2 (HSV-2) increases the risk of HIV-1 acquisition, yet the mechanism for this viral pathogen to regulate the susceptibility of the cervicovaginal mucosa to HIV-1 is virtually unknown. Using ex vivo human ectocervical tissue models, we report greater levels of HIV-1 reverse transcription, DNA integration, RNA expression, and virions release in HIV-1/HSV-2 co-infected tissues compared with HIV-1 only infected tissues (P<0.05). Enhanced HIV-1 replication was associated with increased CD4, CCR5, and CD38 transcription (P<0.05) and increased number of CD4(+)/CCR5(+)/CD38(+) T cells in HIV-1/HSV-2 co-infected tissues compared with tissues infected with HIV-1 alone. Tenofovir (TFV) 1% gel, the leading microbicide candidate, demonstrated only partial protection against HIV-1, when applied vaginally before and after sexual intercourse. It is possible that mucosal inflammation, in particular that induced by HSV-2 infection, may have decreased TFV efficacy. HSV-2 upregulated the number of HIV-1-infected cells and elevated the concentration of TFV needed to decrease HIV-1 infection. Similarly, only high concentrations of TFV inhibited HSV-2 replication in HIV-1/HSV-2-infected tissues. Thus, HSV-2 co-infection and mucosal immune cell activation should be taken into consideration when designing preventative strategies for sexual transmission of HIV-1.

Enhanced HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women correlates with increased inflammatory responses.

Knowledge about early innate immune responses at the mucosal surfaces of the female genital tract is important in understanding the pathogenesis of heterosexual transmission of human immunodeficiency virus type-1 (HIV-1). As estradiol decreases inflammatory responses, we postulated that an estradiol-deficient state such as post-menopause could enhance expression of inflammatory factors that stimulate HIV-1 replication. We compare HIV-1 integration, transcription, and viral p24 release levels among ectocervical tissues obtained from pre- and post-menopausal donors. We detected enhanced HIV-1 p24 release levels in post- compared with pre-menopausal tissues (P<0.0001), but saw no difference in HIV-1 integration. Overall, 100% of post-menopausal tissues exhibited levels of HIV-1 transcription above background compared with only 60% of pre-menopausal tissues. Increased HIV-1 transcription was associated with enhanced interleukin (IL)-1ß, IL-6, monocyte chemotactic protein-1, growth-regulated oncogene-α, and interferon-γ-inducible protein-10 expression. Neutralization and nuclear factor-κB-targeting small-interfering RNA experiments both decreased HIV-1 transcription, suggesting that the early inflammatory response may facilitate HIV-1 replication in ex vivo ectocervical tissues from post-menopausal women.

Effect of tyrosine kinase inhibitors on tyrosine phosphorylation and motility parameters in human sperm.

Tyrosine phosphorylation has recently been associated with capacitation and suggested as a regulator of sperm movement, especially characterizing hyperactivation. The objective of this study was to verify if tyrosine phosphorylation of human sperm proteins was essentially required for the maintenance of motility as well as the development of hyperactivation. Washed sperm were incubated for 6 h in Ham's F10 + 0.35% HSA at 37 degrees C in 5% CO(2), with and without the tyrosine kinase inhibitors genistein, tyrphostin, erbstatin, or herbimycin A and the wide-spectrum kinase inhibitor staurosporin. The concentrations of the inhibitors used in the experiments did not induce sperm toxicity, as measured by membrane integrity and mitochondrial function assays. Samples incubated without the inhibitors (control), increased their tyrosine kinase activity (ELISA), the number and intensity of tyrosine-phosphorylated (PY) protein bands (Western blot), the incidence of PY-immunoreactive sperm (immunofluorescence), and some of the sperm motion characteristics (CASA), such as velocity (VEL), amplitude of lateral head displacement (ALH), and hyperactivation. Among the selective protein tyrosine kinase inhibitors, genistein was the most active and consistent, inhibiting sperm tyrosine kinase activity, PY proteins, incidence of PY sperm, and sperm motility and motion parameters, such as VEL, ALH, and hyperactivation. The rest of the kinase inhibitors decreased motion characteristics to a varied extent and had different effects on phosphorylation parameters. In general, they decreased PY phosphorylation of 2 proteins (83 and 54 kDa) present in whole sperm extracts, and two sets of proteins of low (39-49 kDa) and medium (55-87 kDa) molecular weight present in the Triton X-100-solubilized sperm protein fraction. This inhibition was evident regardless of the total tyrosine kinase activity of the samples or the incidence of PY-immunoreactive sperm. The described findings further support the association between motility and protein tyrosine phosphorylation in human sperm and point to certain proteins as the main linkers.

NF-kappaB cis-acting motifs of the human immunodeficiency virus (HIV) long terminal repeat regulate HIV transcription in human macrophages.

The role of NF-kappaB in the reactivation of human immunodeficiency virus (HIV) from latency in CD4 T lymphocytes is well documented. However, its role in driving HIV transcription in human macrophages, which contain a constitutive nuclear pool of NF-kappaB, is less well understood. In this study we have investigated the role that the constitutive pool of NF-kappaB and the NF-kappaB cis-acting motifs of the HIV long terminal repeat (LTR) play in regulating HIV transcription in human monocytic cells and primary macrophages. Inhibition of the constitutive nuclear pool of NF-kappaB (RelA and RelB) in the promonocytic U937 cell line using dominant-negative IkappaBalpha significantly decreases HIV replication. Moreover, it is demonstrated that in the differentiated monocytic cell line THP1, which contains a constitutive nuclear pool of NF-kappaB (RelB),an HIV provirus containing mutations of the kappaB cis-acting sites in the LTR is transcriptionally impaired. Reduction of the constitutive pool of NF-kappaB in human macrophages by an adenovirus vector expressing a dominant-negative IkappaBalpha also reduces HIV transcription. Lastly, mutation of the NF-kappaB cis-acting sites in the LTR of an R5 HIV provirus completely abrogates the first cycle of HIV transcription. These studies indicate that the cis-acting NF-kappaB motifs of the HIV LTR are critical in initiating HIV transcription in human macrophages and suggest that the constitutive nuclear pool of NF-kappaB is important in regulating HIV transcription in these cells.

The signal transduction pathway of CD23 (Fc epsilon RIIb) targets I kappa B kinase.

Alveolar macrophages play a crucial role in initiating the inflammatory response in allergic asthma through the cross-linking of the low affinity IgE receptors (Fc epsilon RIIb or CD23) by IgE-allergen immunocomplexes. We have previously shown that CD23 cross-linking in monocytes and U937 cells targets I kappa B alpha, leading to the activation of the transcription factor NF-kappa B. We demonstrate in this paper that CD23-initiated signaling in U937 cells leads to hyperphosphorylation of I kappa B alpha at Ser32/Ser36 residues. Overexpression of a dominant-negative I kappa B alpha transgene containing mutations at Ser32/Ser36 completely inhibits degradation of I kappa B alpha, NF-kappa B activation, and gene transcription that follows CD23 cross-linking. Investigation of the second messengers mediating the CD23-dependent activation of NF kappa B demonstrates that I kappa B kinases (IKKs) but not p90rsk are selectively activated following CD23 cross-linking and mediates the phosphorylation of I kappa B alpha. Cotransfection experiments with an IKK beta negative dominant completely inhibit CD23 induced NF kappa B activation. Furthermore, the activation of tyrosine kinase(s) by CD23 is required for the induction of IKK activity, I kappa B alpha degradation, and NF-kappa B nuclear translocation. Taken together, our results show that CD23 cross-linking in the monocytic lineage induces tyrosine kinase activation followed by activation of IKK, which phosphorylates I kappa B alpha at the N-terminal domain (Ser32/Ser36), inducing its degradation, NF-kappa B activation and gene transcription.

Serine 32 and serine 36 of IkappaBalpha are directly phosphorylated by protein kinase CKII in vitro.

IkappaBalpha is an inherently unstable protein which binds to and retains the ubiquitous transcription factor NFkappaB in the cytoplasm of resting cells. A continuous low level translocation of NFkappaB to the nucleus, secondary to the basal turnover of IkappaBalpha, is hypothesized to be necessary for cellular maturation, survival and, potentially, transformation. In response to cellular stimulation by inflammatory cytokines or mitogens, IkappaBalpha is rapidly degraded allowing larger pools of NFkappaB to translocate to the nucleus. Phosphorylation of IkappaBalpha at serine 32 (S32) and serine 36 (S36) is necessary for this stimuli-induced degradation. IKKalpha/beta kinases and p90(rsk1)are involved in stimuli-induced targeting of one or both of these IkappaBalpha sites. Whether other kinases phosphorylate S32 and S36 directly, and if so, what function they serve in NFkappaB activation remains unknown. Here we present evidence of a direct phosphorylation of IkappaBalpha at both S32 and S36 by purified or immunoprecipitated protein kinase CKII (PK-CKII) and a specific in vivo association between IkappaBalpha and PK-CKII. This PK-CKII-specific kinase activity is not found within the IKKalpha/beta-containing signalsome complex and is biochemically distinct from that of the IKKalpha/beta kinases. The identification of an additional N-terminal IkappaBalpha kinase which is constitutively active and not significantly inducible raises numerous possibilities as to its role in cellular function.

Ikappakappa mediates NF-kappaB activation in human immunodeficiency virus-infected cells.

Human monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV) type-1. Persistent HIV infection of these cells results in increased levels of NF-kappaB in the nucleus secondary to increased IkappaBalpha, IkappaBbeta, and IkappaBepsilon degradation, a mechanism postulated to regulate viral persistence. To characterize the molecular mechanisms regulating HIV-mediated degradation of IkappaB, we have sought to identify the regulatory domains of IkappaBalpha targeted by HIV infection. Using monocytic cells stably expressing different transdominant molecules of IkappaBalpha, we determined that persistent HIV infection of these cells targets the NH2 but not the COOH terminus of IkappaBalpha. Further analysis demonstrated that phosphorylation at S32 and S36 is necessary for HIV-dependent IkappaBalpha degradation and NF-kappaB activation. Of the putative N-terminal IkappaBalpha kinases, we demonstrated that the Ikappakappa complex, but not p90(rsk), is activated by HIV infection and mediates HIV-dependent NF-kappaB activation. Analysis of viral replication in cells that constitutively express IkappaBalpha negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-kappaB (p65/p50) nuclear translocation and degree of viral persistence in human monocytes.

Type of rectal contents and infectivity of domiciliary populations of Triatoma infestans (Hemiptera: Reduviidae) in Argentina.

Different types of rectal content in domiciliary populations of Triatoma infestans (Hemiptera: Reduviidae: Triatominae) were associated with differences in the degree of infection with Trypanosoma (Schizotrypanum) cruzi. Field studies were carried out in a region of Argentina (Cordoba) where Chagas' disease is endemic. The rectal contents of domiciliary T. infestans were classified by color (clear, yellow, or brown) and the number of infective metacyclic stages of T. cruzi. The seasonal percentage of triatomines with metacyclics did not vary significantly, except in adults, in which it was greatest in spring (November-December). The mean density of T. cruzi per microliter of rectal material varied during the year, according to instar and color of the rectal contents. Clear recta had most metacyclics. December (late spring) may be more important for vectorial transmission of T. cruzi, because there are more infective adults (788) with higher counts of rectal metacyclics.

Development of Trypanosoma cruzi in Triatoma infestans: influence of temperature and blood consumption.

This work had 2 objectives. The first was to quantify Trypanosoma cruzi development within Triatoma infestans maintained at 2 different temperatures, using an experimental design that simulated the natural transmission process and, second, to learn how the vector blood consumption rate modifies the parasite's development. Two hundred and three, fifth-stage nymphs of T. infestans were infected with the X-1 strain of T. cruzi (about 10(4) trypanosomes/ml blood), maintained at 20 and 28 C, and daily offered the opportunity to feed on uninfected laboratory mice. From 24 hr to the 55th day after the infective meal, the total number of epimastigotes and rectal and fecal metacyclic trypomastigotes were counted. Epimastigote multiplication began on the first day after the infective meal at both temperatures. This parasitic stage developed similar population densities within the vector under both temperature regimes. Trypomastigotes appeared in the rectum and feces at 20 C, 32 and 24 days later, respectively, than at 28 C; however, once they became infective, insects developed similar population densities of fecal metacyclic forms. Blood consumption was related to epimastigote and rectal trypomastigote development at 28 C, but not to the number of trypomastigotes in the feces. A minimum of 120 and 180 mg of fresh blood consumed assured that all bugs developed epimastigotes and trypomastigotes. In spite of the delay in producing metacyclic forms at 20 C, the insect's infective capacity was similar at both temperatures.

Influence of mating on ovarian follicle development in Triatoma infestans (Klug, 1834)

This works examines the influence of mating on ovarian follicle development in Triatoma infestans. The observations were carried out on both virgin and mated females, wich were killed at various times after their emergence. There was no difference in the ovarian development of both experimental groups during the first gonadotrofic cycle. By the 7th day mated females as well as virgn females showed vitellogenic oocytes. The coriogenesis and ovulation process began on the 13th day after imaginal moulting. However we could observe that egg-laying was dependent on mating. Mated females laid eggs whereas virgin females did not lay eggs. However ovarian production was significantly greater in the mated females. It is suggested that in T. infestans mating stimulates egg-laying but it does not influence the oogenesis and ovulation process

Influence of mating on ovarian follicle development in Triatoma infestans (Klug, 1834).

This work examines the influence of mating on ovarian follicle development in Triatoma infestans. The observations were carried out on both virgin and mated females, which were killed at various times after their emergence. There was no difference in the ovarian development of both experimental groups during the first gonadotrophic cycle. By the 7th day mated females as well as virgin females showed vitellogenic oocytes. The coriogenesis and ovulation process began on the 13th day after imaginal moulting. However we could observe that egg-laying was dependent on mating. Mated females laid eggs whereas virgin females did not lay eggs. However ovarian production was significantly greater in the mated females. It is suggested that in T. infestans mating stimulates egg-laying but it does no influence the oogenesis and ovulation process.

Are dead Triatoma infestans a competent vector of Trypanosoma cruzi?

After Triatoma infestans death, Trypanosoma cruzi survived several days, maintaining the ability to infect a vertebrate host. Dead bugs from an endemic area collected during an official spraying comapign showed mobile rectal tripanosomes up to 14 days after vector death. Two days after vector death2, 760 tripomastigotes were found alive in its rectal material. However, the number of mobile tripomastigotes decreased significantly from the 5th day after death. Laboratory proofs with third and fifth nymphal stage showed similar results. Living tripanosomes were found in their rectal material at 10 days in third stage and even at 30 days in fifth nymphal stage. The mean number of tripomastigotes had no changes up to 10 days in third nymphal stage and increased significantly from 1 to 10 days in the fifth stage. Conjuctival instillation as well as intraperitoneal innoculation to mice, of metacyclic forms from dead T. infestans produced infection in the vertebrate host. Present results show that human contact with dead vector highly probable in summer and living and infective T. cruzi are available for transmision in the vector

Are dead Triatoma infestans a competent vector of Trypanosoma cruzi?

After Triatoma infestans death, Trypanosoma cruzi survived several days, maintaining the ability to infect a vertebrate host. Dead bugs from an endemic area collected during an official spraying campaign showed mobile rectal trypanosomes up to 14 days after vector death. Two days after vector death 2,760 trypomastigotes were found alive in its rectal material. However, the number of mobile trypomastigotes decreased significantly from the 5th day after death. Laboratory proofs with third and fifth nymphal stage showed similar results. Living trypanosomes were found in their rectal material at 10 days in third stage and even at 30 days in fifth nymphal stage. The mean number of trypomastigotes had no changes up to 10 days in third nymphal stage and increased significantly from 1 to 10 days in the fifth stage. Conjunctival instillation as well as intraperitoneal inoculation to mice, of metacyclic forms from dead T. infestans produced infection in the vertebrate host. Present results show that human contact with dead vector is highly probable in summer and living and infective T. cruzi are available for transmission in the vector.

Seasonal changes in infectivity of domestic populations of Triatoma infestans.

This paper examines the infection rate of Trypanosoma cruzi in Triatoma infestans, the main vector of Chagas disease in Argentina and neighbouring countries. The study was carried out in 1986-1987 on 5 houses (ranchos) in the endemic area of central Argentina. Domestic T. infestans populations were sampled in each season with a constant capture effort (2.5 man-hours/house) using a chemical irritant. The rectal content of the bugs was examined for the presence of T. cruzi. The vector population density showed seasonal changes with highest values during the hot season (November-April). The percentage of infected bugs was higher in mid-spring (November) and autumn (April) than in winter (August) and early spring (October). The mean number of parasites (epimastigotes and trypomastigotes) per microliter of rectal material was very high during mid- and late spring (December). The percentage and number of metacyclic forms differed between seasons, reaching the highest values in late spring. The percentage of infected bugs in houses with children younger than 10 years old was higher than that in houses without children, during all the seasons. Late spring seemed to be the period when domestic populations of T. infestans had the highest vector potential.