Prevention of recurrent herpes labialis outbreaks through low-intensity laser therapy: a clinical protocol with 3-year follow-up

Conselho Nacional de Pesquisa e Desnsenvolvimento Tecnologico (CNPq)

Genital morphology of the male South American fur seal (Arctocephalus australis) and biological implications

Male capacity for spreading genes to a great number of descendents and to determine population dynamics depend directly on the genital organs. Morphological studies in pinnipeds are scarce and the functional meaning of some characteristics has never been discussed. We hypothesized that Arctocephalus australis (A. australis) shows morphophysiological adaptations in order to guarantee the perpetuation of the species in the unique annual mating season. Seven males, dead from natural causes, had their genital organs collected and fixed for morphological description. Some features differ from other described mammalian males and are closely related to the biology and reproductive cycle of this species, as the scrotal epidermis, absence of glandular portion in the ductus deferens and spermatogenic epithelium suggest a recrudescent testis period. The corona glandis exhibits a singular arrangement: its erectile border looks like a formation of petals and its association with the os penis gives a "lily-flower" form to this region. We propose the name margo petaliformis to this particular erectile border of the corona glandis because of its similarity to a flower corola. The male genital organs of A. australis show morphological features compatible with adaptation to environment requirements and reproductive efficiency.

Meningoencefalite concomitante a herpes zoster oftálmico em escolar previamente hígido

INTRODUÇÃO O vírus Varicella-Zoster é o agente da varicela, doença auto-limitada comum da faixa etária pediátrica. Cerca de 20% dos casos evoluem com herpes zoster em algum momento da vida, devido a reativação do vírus dos gânglios nervosos ou reexposição. O envolvimento do ramo oftálmico do nervo trigêmio, defido como zoster oftálmico, tem como complicação mais descrita a nevralgia pós-herpética, podendo evoluir com outras alterações locais agudas e tardias. Meningoencefalites concomitante a herpes zoster são pouco descritas na literatura. DESCRIÇÃO DO CASO Paciente de 9 anos, com antecedente de varicela com 1 ano de idade, com queixa de vômitos e cefaléia há dois dias, associada a queda do estado gerale hiporexia. Referiu aparecimento de lesões vesico-bolhosas dolorosas em região periorbitária esquerda há 1 dia e evoluiu com agitação psicomotora e confusão mental. No exame físico de entrada apresentava-se sonolento, com lesões vesico-bolhosas em dermátomo do ramo oftálmico do nervo trigêmio, sem sinais de irritação meníngea ou déficits motores, sem alterações visuais ou oculares. Realizada tomografia de crânio e eletroencefalograma sem alterações. Coletado líquor que revelou líquido límpido e incolor, com aumento de celularidade às custas de linfócitos, glicorraquia normal, bacterioscopia negativa e culturas negativas. Feita hipótese diagnóstica inicial de herpes zoster oftálmico complicado com meningoencefalite e introduzido aciclovir. Paciente evoluiu bem, com melhora do estado geral, remissão dos sintomas neurológicos e melhora das lesões de pele. Evidenciado PCR positivo para o vírus varicela-zoster (VVZ) no líquor. DISCUSSÃO Encontramos poucas descrições na literatura de herpes zoster oftálmico associado à alterações neurológicas. A presença da PCR positiva no liquor foi fundamental para o diagnóstico. CONCLUSÃO O VVZ pode reativar na forma de herper zoster oftálmico e acometer o sistema nervoso central. Apesar de evento raro em crianças, especialmente nas imunocompetentes, a presença da PCR positiva liquor confirmou a meningoencefalite.

Morphology of accessory genital glands of spotted paca(Agouti paca Linnaeus, 1766)

The spotted paca is the second largest rodent in Brazil, where it is of great economic interestin impoverished regions in view of its prominence as a low-cost source of protein. Littleis known about the morphology of the accessory genital glands of this species. Thus, westudied the position and morphology of the genitals in ten adult male spotted pacas. Theanimals were divided into two groups, five animals were used for fixing of samples in 10%aqueous formaldehyde for macroscopic studies and the other five animals were designatedfor microscopic analysis. These were arranged in pairs and had the vesicular, prostate,coagulating and bulbourethral glands identified, being structured as mucous glands, whichlead into the pelvic urethra. It was concluded that the accessory genital glands found in thepaca are the same as those found in most rodents, showing similar histological aspects

Interazioni tra le glicoproteine D, B, H ed L critiche per la fusione indotta dal virus Herpes simplex

Four glycoproteins (gD, gB, gH, and gL) are required for herpes simplex virus (HSV) entry into the cell and for cell-cell fusion in transfected cells. gD serves as the receptor-binding glycoprotein and as the trigger of fusion; the other three glycoproteins execute fusion between the viral envelope and the plasma or endocytic membranes. Little is known on the interaction of gD with gB, gH, and gL. Here, the interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. Split EGFP complementation was detected between proteins designated gDN + gHC, gDN + gBC, and gHN + gBC + wtgD, both in cells transfected with two or tree glycoproteins and in cells transfected with the four glycoproteins, commited to form syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently one of the other. We further document the interaction between gH and gB. To elucidate which portions of the glycoproteins interact with each other we generated mutants of gD and gB. gD triggers fusion through a specialised domain, named pro-fusion domain (PFD), located C-terminally in the ectodomain. Here, we show that PFD is made of subdomains 1 and 2 (amino acids 260–285 and 285–310) and that each one partially contributed to herpes simplex virus infectivity. Chimeric gB molecules composed of HSV and human herpesvirus 8 (HHV8) sequences failed to reach the cell surface and to complement a gB defective virus. By means of pull down experiments we analyzed the interactions of HSV-HHV8 gB chimeras with gH or gD fused to the strep-tag. The gB sequence between aa residues 219-360 was identified as putative region of interaction with gH or critical to the interaction.

Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry.

Interazioni tra Glicoproteine che mediano la penetrazione del Virus Herpes Simplex nella cellula e disegno razionale di Peptidi Inibitori

Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of gD, gB and heterodimer gH/gL. gD serves as receptor-binding glycoprotein and trigger of fusion; its ectodomain is organized in a N-terminal domain carrying the receptor-binding sites, and a C-terminal domain carrying the profusion domain, required for fusion but not receptor-binding. gB and gH/gL execute fusion. To understand how the four glycoproteins cross-talk to each other we searched for biochemical defined complexes in infected and transfected cells, and in virions. We report that gD formed complexes with gB in absence of gH/gL, and with gH/gL in absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry, and did not increase at virus fusion with cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the aa 240-260 portion and downstream, with T306P307 as critical residues, and of the binding site to gH/gL at aa 260-310 portion, with P291P292 as critical residues. The results indicate that gD carries composite independent binding sites for gB and gH/gL, both of which partly located in the profusion domain. The second part of the project dealt with rational design of peptides inhibiting virus entry has been performed. Considering gB and gD, the crystal structure is known, so we designed peptides that dock in the structure or prevent the adoption of the final conformation of target molecule. Considering the other glycoproteins, of which the structure is not known, peptide libraries were analyzed. Among several peptides, some were identified as active, designed on glycoprotein B. Two of them were further analyzed. We identified peptide residues fundamental for the inhibiting activity, suggesting a possible mechanism of action. Furthermore, changing the flexibility of peptides, an increased activity was observed,with an EC50 under 10μM. New approaches will try to demonstrate the direct interaction between these peptides and the target glycoprotein B.

Genetic engineering and preclinical evaluation of oncolytic herpes simplex viruses retargeted to cancer-specific receptors

Oncolytic virotherapy exploits the ability of viruses to infect and kill cells. It is suitable as treatment for tumors that are not accessible by surgery and/or respond poorly to the current therapeutic approach. HSV is a promising oncolytic agent. It has a large genome size able to accommodate large transgenes and some attenuated oncolytic HSVs (oHSV) are already in clinical trials phase I and II. The aim of this thesis was the generation of HSV-1 retargeted to tumor-specific receptors and detargeted from HSV natural receptors, HVEM and Nectin-1. The retargeting was achieved by inserting a specific single chain antibody (scFv) for the tumor receptor selected inside the HSV glycoprotein gD. In this research three tumor receptors were considered: epidermal growth factor receptor 2 (HER2) overexpressed in 25-30% of breast and ovarian cancers and gliomas, prostate specific membrane antigen (PSMA) expressed in prostate carcinomas and in neovascolature of solid tumors; and epidermal growth factor receptor variant III (EGFRvIII). In vivo studies on HER2 retargeted viruses R-LM113 and R-LM249 have demonstrated their high safety profile. For R-LM249 the antitumor efficacy has been highlighted by target-specific inhibition of the growth of human tumors in models of HER2-positive breast and ovarian cancer in nude mice. In a murine model of HER2-positive glioma in nude mice, R-LM113 was able to significantly increase the survival time of treated mice compared to control. Up to now, PSMA and EGFRvIII viruses (R-LM593 and R-LM613) are only characterized in vitro, confirming the specific retargeting to selected targets. This strategy has proved to be generally applicable to a broad spectrum of receptors for which a single chain antibody is available.

Human herpes virus and Alzheimer’s disease

Alzheimer’s disease (AD) is a chronic and progressive neurodegenerative disorder and according to the WHO it is estimated that 36 millions of people worldwide currently suffer from AD. Genetic and environmental factors interact in a complex interplay that might affect pathogenic mechanisms leading to age-related neurodegeneration. The hypothesis is that the presence of allelic polymorphisms in selected genes affecting individual brain susceptibility to infection by the herpes virus family during aging, may contribute to neuronal loss, inflammation and amyloid deposition. Herpes virus family show features relevant to AD, since they infect a large proportion of human population, develop a latent form persisting for several years, are difficult to eliminate by immune responses especially when latency has been established and are able to infect neurons. The association between AD and herpes viruses infection has been investigated. In particular the investigation focused on CMV, EBV and HHV-6 in DNA samples from peripheral blood of a large cohort of patients with clinical diagnosis of AD and age matched CTR, from a longitudinal population study, and DNA samples from brain tissue of patients with neuropathological diagnosis of definitive AD. An association between the presence of EBV and HHV-6 DNA from PBL positivity with the cognitive deterioration and progression to AD has been focused. Moreover, IgG plasma levels in CTR and AD to these viruses were tested. CMV and EBV IgG plasma levels were higher in elderly subjects that developed clinical AD at the end of the five year follow up. Our findings support the notion that persistent cycles of latency and reactivation of herpes viruses may contribute to impair systemic immune response and induce altered inflammatory process that in turn affect cognitive decline during aging.

Role of αvβ3 – Integrin and TLR2 in the innate response to Herpes Simplex Virus infection and Delivery of retargeted oncolytic Herpes Simplex via carrier cells

In the first part of my thesis I studied the mechanism of initiation of the innate response to HSV-1. Innate immune response is the first line of defense set up by the cell to counteract pathogens infection and it is elicited by the activation of a number of membrane or intracellular receptors and sensors, collectively indicated as PRRs, Patter Recognition Receptors. We reported that the HSV pathogen-associated molecular patterns (PAMP) that activate Toll-like receptor 2 (TLR2) and lead to the initiation of innate response are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the Herpesvirus. Specifically gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation. Then, by gain and loss-of-function approaches, we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against HSV-1. We showed that αvβ3-integrin signals through a pathway that concurs with TLR2, affects activation/induction of interferons type 1, NF-κB, and a polarized set of cytokines and receptors. Thus, we demonstrated that gH/gL is sufficient to induce IFN1 and NF-κB via this pathway. From these data, we proposed that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors. In the second part of my thesis I studied the capacity of human mesenchymal stromal cells isolated by fetal membranes (FM-hMSCs) to be used as carrier cells for the delivery of retargeted R-LM249 virus. The use of systemically administrated carrier cells to deliver oncolytic viruses to tumoral targets is a promising strategy in oncolytic virotherapy. We observed that FM-hMSCs can be infected by R-LM249 and we optimized the infection condition; then we demonstrate that stromal cells sustain the replication of retargeted R-LM249 and spread it to target tumoral cells. From these preliminary data FM-hMSCs resulted suitable to be used as carrier cells

Screening and treatment to prevent sequelae in women with Chlamydia trachomatis genital infection: how much do we know?

Background. An important question for chlamydia control programs is the extent to which finding and treating prevalent, asymptomatic Chlamydia trachomatis genital infection reduces reproductive sequelae in infected women. Methods. We reviewed the literature to critically evaluate evidence on the effect of chlamydia screening on development of sequelae in infected women. Results. Two randomized controlled trials of 1-time screening for chlamydial infection—in a Seattle-area health maintenance organization and a Danish school district—revealed that screening was associated with an ∼50% reduction in the incidence of pelvic inflammatory disease over the following year. However, both of these trials had methodological issues that may have affected the magnitude of observed screening benefits and might limit generalizability to other populations. A large, nonrandomized cohort of chlamydia screening among US Army recruits, although limited by lack of outpatient data, did not find a benefit of similar magnitude to the randomized trials. Methodological limitations restrict valid conclusions about individual benefits of screening using data from historical cohorts and ecological studies. We identified no trials directly evaluating the effect of chlamydia screening on subclinical tubal inflammation or damage, ectopic pregnancy, or tubal factor infertility and no studies addressing the effects of >1 round of screening, the optimal frequency of screening, or the benefits of screening for repeat infections. Conclusions. Additional studies of the effectiveness of chlamydia screening would be valuable; feasible study designs may depend on the degree to which screening programs are already established. In addition, better natural history data on the timing of tubal inflammation and damage after C. trachomatis infection and development of more accurate, noninvasive tools to assess chlamydial sequelae are essential to informing chlamydia control efforts.

Risk of sequelae after Chlamydia trachomatis genital infection in women

Chlamydia trachomatis infection, the most common reportable disease in the United States, can lead to pelvic inflammatory disease (PID), infertility, ectopic pregnancy, and chronic pelvic pain. Although C. trachomatis is identified among many women who receive a diagnosis of PID, the incidence and timing of PID and long-term sequelae from an untreated chlamydial infection have not been fully determined. This article examines evidence reviewed as part of the Centers for Disease Control and Prevention Chlamydia Immunology and Control Expert Advisory Meeting; 24 reports were included. We found no prospective studies directly assessing risk of long-term reproductive sequelae, such as infertility, after untreated C. trachomatis infection. Several studies assessed PID diagnosis after untreated chlamydial infection, but rates varied widely, making it difficult to determine an overall estimate. In high-risk settings, 2%-5% of untreated women developed PID within the approximately 2-week period between testing positive for C. trachomatis and returning for treatment. However, the rate of PID progression in the general, asymptomatic population followed up for longer periods appeared to be low. According to the largest studies, after symptomatic PID of any cause has occurred, up to 18% of women may develop infertility. In several studies, repeated chlamydial infection was associated with PID and other reproductive sequelae, although it was difficult to determine whether the risk per infection increased with each recurrent episode. The present review critically evaluates this body of literature and suggests future research directions. Specifically, prospective studies assessing rates of symptomatic PID, subclinical tubal damage, and long-term reproductive sequelae after C. trachomatis infection; better tools to measure PID and tubal damage; and studies on the natural history of repeated chlamydial infections are needed.

"Cutting for love": genital incisions to enhance sexual desirability and commitment in KwaZulu-Natal, South Africa

Several studies have documented women's use of vaginal practices in South Africa to enhance their desirability to men. This article describes a little known practice of this kind among women in KwaZulu-Natal. It involves the use of small incisions in the genital area (and often abdomen and breasts) to introduce herbal substances, described as love medicines, into the body through the incisions. In-depth interviews were carried out with 20 key informants and 20 women, and eight focus group discussions with women and men, in a rural and urban site in 2005-06. A province-wide household survey was then conducted using a multi-stage cluster sample design among 867 women aged 18-60. Forty-two per cent of the women in the household survey had heard of genital incisions; only 3% had actually used them. The main motivation was the enhancement of sexual attractiveness and long-term partner commitment. It appears to be a very recent practice, but may be an extension of an older healing practice not involving the genitals. It was most prevalent among rural women aged 24-29 (although not significant), those with less education, and those who suspected their partners of having other partners. It is linked to the modern popularity of love medicines, which in turn illustrates the troubling state of gender relations in KwaZulu-Natal today.

[Laparoscopic management of genital prolapse by lateral suspension using mesh: a series of 377 patients]

Genital prolapse is frequent and can be found in about 50% of parous women. Its etiology is complex and multifactorial. Predisposing factors include: genetics (connective tissue disorders, family history); general state (age, parity, weight, smoking, obstructive pulmonary disease); trauma (carrying heavy loads, intense physical exercise); or iatrogenic (post hysterectomy). Treatment can be conservative or surgical and depends mainly on the severity of symptoms. Developments in surgical techniques and synthetic material in the last 20 years enabled us to use minimally invasive procedures with improved post operative course and decreased recurrence rates.