Regular Use of Depot Medroxyprogesterone Acetate Causes Thinning of the Superficial Lining and Apical Distribution of Human Immunodeficiency Virus Target Cells in the Human Ectocervix.

BACKGROUND: The hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may be associated with an increased risk of acquiring human immunodeficiency virus (HIV). We hypothesize that DMPA use influences the ectocervical tissue architecture and HIV target cell localization. METHODS: Quantitative image analysis workflows were developed to assess ectocervical tissue samples collected from DMPA users and control subjects not using hormonal contraception. RESULTS: Compared to controls, the DMPA group exhibited a significantly thinner apical ectocervical epithelial layer and a higher proportion of CD4+CCR5+ cells with a more superficial location. This localization corresponded to an area with a nonintact E-cadherin net structure. CD4+Langerin+ cells were also more superficially located in the DMPA group, although fewer in number compared to the controls. Natural plasma progesterone levels did not correlate with any of these parameters, whereas estradiol levels were positively correlated with E-cadherin expression and a more basal location for HIV target cells of the control group. CONCLUSIONS: DMPA users have a less robust epithelial layer and a more apical distribution of HIV target cells in the human ectocervix, which could confer a higher risk of HIV infection. Our results highlight the importance of assessing intact genital tissue samples to gain insights into HIV susceptibility factors.

The Feasibility of Host Transcriptome Profiling as a Diagnostic Tool for Microbial Etiology in Childhood Cancer Patients with Febrile Neutropenia.

Infection is a common and serious complication of cancer treatment in children that often presents as febrile neutropenia (FN). Gene-expression profiling techniques can reveal transcriptional signatures that discriminate between viral, bacterial and asymptomatic infections in otherwise healthy children. Here, we examined whether gene-expression profiling was feasible in children with FN who were undergoing cancer treatment. The blood transcriptome of the children (n = 63) was investigated at time of FN diagnosed as viral, bacterial, co-infection or unknown etiology, respectively, and compared to control samples derived from 12 of the patients following the FN episode. RNA sequencing was successful in 43 (68%) of the FN episodes. Only two genes were significantly differentially expressed in the bacterial versus the control group. Significantly up-regulated genes in patients with the other three etiologies versus the control group were enriched with cellular processes related to proliferation and cellular stress response, with no clear enrichment with innate responses to pathogens. Among the significantly down-regulated genes, a few clustered into pathways connected to responses to infection. In the present study of children during cancer treatment, the blood transcriptome was not suitable for determining the etiology of FN because of too few circulating immune cells for reliable gene expression analysis.

The neovaginal microbiome of transgender women post-gender reassignment surgery.

BACKGROUND: Gender reassignment surgery is a procedure some transgender women (TW) undergo for gender-affirming purposes. This often includes the construction of a neovagina using existing penile and scrotal tissue and/or a sigmoid colon graft. There are limited data regarding the composition and function of the neovaginal microbiome representing a major gap in knowledge in neovaginal health. RESULTS: Metaproteomics was performed on secretions collected from the neovaginas (n = 5) and rectums (n = 7) of TW surgically reassigned via penile inversion/scrotal graft with (n = 1) or without (n = 4) a sigmoid colon graft extension and compared with secretions from cis vaginas (n = 32). We identified 541 unique bacterial proteins from 38 taxa. The most abundant taxa in the neovaginas were Porphyromonas (30.2%), Peptostreptococcus (9.2%), Prevotella (9.0%), Mobiluncus (8.0%), and Jonquetella (7.2%), while cis vaginas were primarily Lactobacillus and Gardnerella. Rectal samples were mainly composed of Prevotella and Roseburia. Neovaginas (median Shannon's H index = 1.33) had higher alpha diversity compared to cis vaginas (Shannon's H = 0.35) (p = 7.2E-3, Mann-Whitney U test) and were more similar to the non-Lactobacillus dominant/polymicrobial cis vaginas based on beta diversity (perMANOVA, p = 0.001, r2 = 0.342). In comparison to cis vaginas, toll-like receptor response, amino acid, and short-chain fatty acid metabolic pathways were increased (p < 0.01), while keratinization and cornification proteins were decreased (p < 0.001) in the neovaginal proteome. CONCLUSIONS: Penile skin-lined neovaginas have diverse, polymicrobial communities that show similarities in composition to uncircumcised penises and host responses to cis vaginas with bacterial vaginosis (BV) including increased immune activation pathways and decreased epithelial barrier function. Developing a better understanding of microbiome-associated inflammation in the neovaginal environment will be important for improving our knowledge of neovaginal health. Video Abstract.

The Role of TPMT, ITPA, and NUDT15 Variants during Mercaptopurine Treatment of Swedish Pediatric Patients with Acute Lymphoblastic Leukemia.

OBJECTIVE: To evaluate the roles of thiopurine methyltransferase (TPMT), inosine triphosphatase (ITPA), and Nudix hydrolase 15 (NUDT15) in 6-mercaptopurine (6-MP) sensitivity during treatment of pediatric patients with acute lymphoblastic leukemia (ALL). STUDY DESIGN: The study included 102 pediatric patients with ALL subject to the Nordic society Of Paediatric Haematology and Oncology (NOPHO) ALL-2000 and ALL-2008 protocols. Episodes of neutropenia and febrile neutropenia, TPMT sequence variants, as well as 6-MP end doses, were collected retrospectively from medical records. TPMT, ITPA, and NUDT15 sequence variants were analyzed using pyrosequencing. RESULTS: TPMT variants were associated with a reduced risk of neutropenia and febrile neutropenia during the maintenance II period (P = .019 and P < .0001, respectively). In addition, a NUDT15 variant was associated with a lower end dose of 6-MP (P = .0097), but not with neutropenia and febrile neutropenia. ITPA variants were not associated with an increased risk of neutropenia, febrile neutropenia, nor lower end dose of 6-MP. However, when analyzing the entire treatment period, ITPA variants were associated with a decreased risk of febrile neutropenia. CONCLUSIONS: White blood cell count-based dose adjustments are regularly performed for known TPMT- deficient patients and results in a reduced risk of neutropenia and febrile neutropenia. Also in NUDT15-deficient patients dose adjustments are performed as indicated by low end dose of 6-MP. ITPA-deficient patients had a decreased risk of febrile neutropenia when analyzing the entire treatment period. Our data suggest that NUDT15 plays an important role in 6-MP treatment and the results should be confirmed in larger cohorts. Future studies should also follow up whether white blood cell count-based dose adjustments affect the risk of relapse.

Impact of Q-Griffithsin anti-HIV microbicide gel in non-human primates: In situ analyses of epithelial and immune cell markers in rectal mucosa.

Natural-product derived lectins can function as potent viral inhibitors with minimal toxicity as shown in vitro and in small animal models. We here assessed the effect of rectal application of an anti-HIV lectin-based microbicide Q-Griffithsin (Q-GRFT) in rectal tissue samples from rhesus macaques. E-cadherin+ cells, CD4+ cells and total mucosal cells were assessed using in situ staining combined with a novel customized digital image analysis platform. Variations in cell numbers between baseline, placebo and Q-GRFT treated samples were analyzed using random intercept linear mixed effect models. The frequencies of rectal E-cadherin+ cells remained stable despite multiple tissue samplings and Q-GRFT gel (0.1%, 0.3% and 1%, respectively) treatment. Whereas single dose application of Q-GRFT did not affect the frequencies of rectal CD4+ cells, multi-dose Q-GRFT caused a small, but significant increase of the frequencies of intra-epithelial CD4+ cells (placebo: median 4%; 1% Q-GRFT: median 7%) and of the CD4+ lamina propria cells (placebo: median 30%; 0.1-1% Q-GRFT: median 36-39%). The resting time between sampling points were further associated with minor changes in the total and CD4+ rectal mucosal cell levels. The results add to general knowledge of in vivo evaluation of anti-HIV microbicide application concerning cellular effects in rectal mucosa.

A High-throughput Bead-based Affinity Assay Enables Analysis of Genital Protein Signatures in Women At Risk of HIV Infection.

Women at high risk of HIV infection, including sex workers and those with active genital inflammation, have molecular signatures of immune activation and epithelial barrier remodeling in samples of their genital mucosa. These alterations in the local immunological milieu are likely to impact HIV susceptibility. We here analyze host genital protein signatures in HIV uninfected women, with high frequency of condom use, living in HIV-serodiscordant relationships. Cervicovaginal secretions from women living in HIV-serodiscordant relationships (n = 62) were collected at three time points over 12 months. Women living in HIV-negative seroconcordant relationships (controls, n = 25) were sampled at one time point. All study subjects were examined for demographic parameters associated with susceptibility to HIV infection. The cervicovaginal samples were analyzed using a high-throughput bead-based affinity assay. Proteins involved in epithelial barrier function and inflammation were increased in HIV-serodiscordant women. By combining several methods of analysis, a total of five proteins (CAPG, KLK10, SPRR3, elafin/PI3, CSTB) were consistently associated with this study group. Proteins analyzed using the affinity set-up were further validated by label-free tandem mass spectrometry in a partially overlapping cohort with concordant results. Women living in HIV-serodiscordant relationships thus had elevated levels of proteins involved in epithelial barrier function and inflammation despite low prevalence of sexually transmitted infections and a high frequency of safe sex practices. The identified proteins are important markers to follow during assessment of mucosal HIV susceptibility factors and a high-throughput bead-based affinity set-up could be a suitable method for such evaluation.

Increased Cervical CD4+CCR5+ T Cells Among Kenyan Sex Working Women Using Depot Medroxyprogesterone Acetate.

Depot medroxyprogesterone acetate (DMPA) is the most common hormonal contraceptive used by women in sub-Saharan Africa, however, it has been epidemiologically associated with HIV infections. To assess whether DMPA has an effect on the number and activation of HIV target cells, this study assessed the levels and phenotype of blood- and mucosal-derived HIV target cells among women using DMPA. Thirty-five HIV uninfected women from the Pumwani Sex Worker cohort from Nairobi, Kenya were enrolled in the study (15 using DMPA and 20 not using hormonal contraception). Blood (plasma and peripheral blood mononuclear cells) and cervicovaginal (lavage, cervical cells, and ectocervical biopsies) samples were collected. Cellular phenotype and activation status were determined by flow cytometry, cytokine levels were assessed by bead array and image analysis assessed cell number and phenotype in situ. In blood, the proportion of HIV target cells and activated T cells was lower in DMPA users versus those not using hormonal contraceptives. However, analysis of cervical mononuclear cells showed that DMPA users had elevated levels of activated T cells (CD4+CD69+) and expressed lower levels of the HIV co-receptor CCR5 on a per cell basis, while tissue samples showed that in the ectocervix, DMPA users had a higher proportion of CD4+CCR5+ T cells. This study demonstrates that DMPA users had higher levels of activated T cells and HIV target cells in the genital tract. The increased pool of mucosal HIV target cells provides new biological information about the potential impact of DMPA on HIV susceptibility.

Human IgM monoclonal antibodies block HIV-transmission to immune cells in cervico-vaginal tissues and across polarized epithelial cells in vitro.

The importance of natural IgM antibodies in protection against infections is still emerging and these antibodies have a potential role in the maintenance of homeostasis through clearance of apoptotic bodies, complement-dependent mechanisms, inflammation and exclusion of misfolded proteins. Natural IgM act as a first line of defence against unknown hazardous factors and are present in most vertebrates. We investigated the functional capacity of anti-HIV-1 IgM monoclonal antibodies, from a combinatorial Fab library derived from healthy individuals, and evaluated their protective role in inhibiting HIV-1 in vitro when passing across the human mucosal epithelial barrier. Primary HIV-1 isolates were efficiently transmitted over the tight polarized epithelial cells when added to their apical surface. Efficient inhibition of HIV-1 transmission was achieved when anti-HIV-1 IgM monoclonal antibodies were added to the basolateral side of the cells. Two of these human IgM MoAbs had the ability to neutralize HIV and reduced infection of dendritic cells in primary cervico-vaginal tissue biopsies in vitro. This indicates a potential role of natural IgM antibodies in the reduction of HIV-1 transmission in mucosal tissues and improve our understanding of how natural IgM antibodies against a neutralizing epitope could interfere with viral transmission.

The vaginal microbiome amplifies sex hormone-associated cyclic changes in cervicovaginal inflammation and epithelial barrier disruption.

PROBLEM: Susceptibility to HIV is associated with the menstrual cycle and vaginal microbiome, but their collective impact on vaginal inflammation remains unclear. Here, we characterized the cervicovaginal proteome, inflammation, and microbiome community structure and function during the menstrual cycle. METHOD OF STUDY: Cervicovaginal secretions were collected from regularly cycling women (n = 16) at median day 10, 16, and 24 of each menstrual cycle and analyzed by mass spectrometry, 16S rRNA gene sequencing, and a multiplex bead array immunoassay. Follicular, ovulatory, and luteal phases were defined by serum sex hormone levels. RESULTS: Ovulation showed the largest mucosal proteome changes, where 30% and 19% of the 406 human proteins identified differed compared to the luteal and follicular phases, respectively. Neutrophil/leukocyte migration pathways were lowest during ovulation and peaked in the luteal phase, while antimicrobial and epithelial barrier promoting proteins were highest during ovulation. Vaginal microbial community structure and function did not vary significantly during the menstrual cycle, with the majority consistently Lactobacillus-dominant (63%) or non-Lactobacillus-dominant (25%). Fluctuations in the epithelial barrier protein RPTN between the ovulatory and luteal phase were amplified in women with Gardnerella vaginalis and anaerobic bacteria and reduced when Lactobacillus was dominant. CONCLUSION: This small study demonstrates that sex hormones modulate neutrophil/leukocyte inflammation, barrier function, and antimicrobial pathways in the female genital tract with the strongest changes occurring during ovulation. The data further suggest a microbiome context for hormone-driven changes in vaginal immunity which may have implications for HIV susceptibility.

Human Immunodeficiency Virus-Infected Women Have High Numbers of CD103-CD8+ T Cells Residing Close to the Basal Membrane of the Ectocervical Epithelium.

Background: Genital mucosa is the main portal of entry for various incoming pathogens, including human immunodeficiency virus (HIV), hence it is an important site for host immune defenses. Tissue-resident memory T (TRM) cells defend tissue barriers against infections and are characterized by expression of CD103 and CD69. In this study, we describe the composition of CD8+ TRM cells in the ectocervix of healthy and HIV-infected women. Methods: Study samples were collected from healthy Swedish and Kenyan HIV-infected and uninfected women. Customized computerized image-based in situ analysis was developed to assess the ectocervical biopsies. Genital mucosa and blood samples were assessed by flow cytometry. Results: Although the ectocervical epithelium of healthy women was populated with bona fide CD8+ TRM cells (CD103+CD69+), women infected with HIV displayed a high frequency of CD103-CD8+ cells residing close to their epithelial basal membrane. Accumulation of CD103-CD8+ cells was associated with chemokine expression in the ectocervix and HIV viral load. CD103+CD8+ and CD103-CD8+ T cells expressed cytotoxic effector molecules in the ectocervical epithelium of healthy and HIV-infected women. In addition, women infected with HIV had decreased frequencies of circulating CD103+CD8+ T cells. Conclusions: Our data provide insight into the distribution of CD8+ TRM cells in human genital mucosa, a critically important location for immune defense against pathogens, including HIV.

Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants.

The most immediate and evident effect of mucosal exposure to semen in vivo is a local release of proinflammatory mediators accompanied by an influx of leukocytes into the female genital mucosa (FGM). The implication of such response in HIV-1 transmission has never been addressed due to limitations of currently available experimental models. Using human tissue explants from the uterine cervix, we developed a system of mucosal exposure to seminal plasma (SP) that supports HIV-1 replication. Treatment of ectocervical explants with SP resulted in the upregulation of inflammatory and growth factors, including IL-6, TNF, CCL5, CCL20, CXCL1, and CXCL8, and IL1A, CSF2, IL7, PTGS2, as evaluated by measuring protein levels in explant conditioned medium (ECM) and gene expression in tissue. SP treatment was also associated with increased recruitment of monocytes and neutrophils, as observed upon incubation of peripheral blood leukocytes with ECM in a transwell system. To evaluate the impact of the SP-mediated response on local susceptibility to HIV-1, we infected ectocervical explants with the CCR5-tropic variant HIV-1BaL either in the presence of SP, or after explant pre-incubation with SP. In both experimental settings SP enhanced virus replication as evaluated by HIV-1 p24gag released in explant culture medium over time, as well as by HIV-1 DNA quantification in explants infected in the presence of SP. These results suggest that a sustained inflammatory response elicited by SP soon after coitus may promote HIV-1 transmission to the FGM. Nevertheless, ectocervical tissue explants did not support the replication of transmitted/founder HIV-1 molecular clones, regardless of SP treatment. Our system offers experimental and analytical advantages over traditional models of HIV-1 transmission for the study of SP immunoregulatory effect on the FGM, and may provide a useful platform to ultimately identify new determinants of HIV-1 infection at this site.

Genital Injury Signatures and Microbiome Alterations Associated With Depot Medroxyprogesterone Acetate Usage and Intravaginal Drying Practices.

Background: Increasing evidence suggests depot medroxyprogesterone acetate (DMPA) and intravaginal practices may be associated with human immunodeficiency virus (HIV-1) infection risk; however, the mechanisms are not fully understood. This study evaluated the effect of DMPA and intravaginal practices on the genital proteome and microbiome to gain mechanistic insights. Methods: Cervicovaginal secretions from 86 Kenyan women, including self-reported DMPA users (n = 23), nonhormonal contraceptive users (n = 63), and women who practice vaginal drying (n = 46), were analyzed using tandem-mass spectrometry. Results: We identified 473 human and 486 bacterial proteins from 18 different genera. Depot medroxyprogesterone acetate use associated with increased hemoglobin and immune activation (HBD, HBB, IL36G), and decreased epithelial repair proteins (TFF3, F11R). Vaginal drying associated with increased hemoglobin and decreased phagocytosis factors (AZU1, MYH9, PLAUR). Injury signatures were exacerbated in DMPA users who also practiced vaginal drying. More diverse (H index: 0.71 vs 0.45; P = .009) bacterial communities containing Gardnerella vaginalis associated with vaginal drying, whereas DMPA showed no significant association with community composition or diversity. Conclusions: These findings provide new insights into the impact of DMPA and vaginal drying on mucosal barriers. Future investigations are needed to confirm their relationship with HIV risk in women.

Frequent Respiratory Viral Infections in Children with Febrile Neutropenia - A Prospective Follow-Up Study.

OBJECTIVE: Febrile neutropenia is common in children undergoing chemotherapy for the treatment of malignancies. In the majority of cases, the cause of the fever is unknown. Although respiratory viruses are commonly associated with this condition, the etiologic significance of this finding remains unclear and is therefore the subject of this study. STUDY DESIGN: Nasopharyngeal aspirates were collected during 87 episodes of febrile neutropenia in children age 0-18 years, being treated at a children's oncology unit between January 2013 and June 2014. Real-time polymerase chain reaction was used to determine the presence of 16 respiratory viruses. Follow-up samples were collected from children who tested positive for one or more respiratory viruses. Rhinoviruses were genotyped by VP4/VP2 sequencing. Fisher's exact test and Mann-Whitney U test were used for group comparisons. RESULTS: At least one respiratory virus was detected in samples from 39 of 87 episodes of febrile neutropenia (45%), with rhinoviruses the most frequently detected. Follow-up samples were collected after a median of 28 days (range, 9-74 days) in 32 of the 39 virus-positive episodes. The respiratory viral infection had resolved in 25 episodes (78%). The same virus was detected at follow-up in one coronavirus and six rhinovirus episodes. Genotyping revealed a different rhinovirus species in two of the six rhinovirus infections. CONCLUSION: The frequency of respiratory viral infections in this group of patients suggests an etiologic role in febrile neutropenia. However, these findings must be confirmed in larger patient cohorts.

Improving Adherence to Post-Cervical Biopsy Sexual Abstinence in Kenyan Female Sex Workers.

PROBLEM: Cervical biopsies offer a unique opportunity for studying local immune response. To investigate hormonally induced immune fluctuations in cervical tissues of Kenyan female sex workers, we improved biopsy sampling protocol safety. Here, we report on steps taken to minimize exposure to HIV following two cervical biopsies. METHODS OF STUDY: Women were asked to abstain from vaginal intercourse to limit HIV exposure during wound healing with financial compensation. A comprehension tool for informed consent, on-site detection of prostate-specific antigens indicating unprotected intercourse within 48 hr, and bi-weekly text message reminders were implemented. RESULTS: The implemented methods improved compliance with post-procedure abstinence by two times (P = 0.013). Fourteen days following a cervical biopsy, no sign of genital inflammation or change in HIV T-cell target proportion were observed. CONCLUSIONS: This study provides new tools for limiting HIV exposure in studies requiring biopsy sampling among women at risk of acquiring HIV.

In Situ Staining and Laser Capture Microdissection of Lymph Node Residing SIV Gag-Specific CD8+ T cells--A Tool to Interrogate a Functional Immune Response Ex Vivo.

While a plethora of data describes the essential role of systemic CD8+ T cells in the control of SIV replication little is known about the local in situ CD8+ T cell immune responses against SIV at the intact tissue level, due to technical limitations. In situ staining, using GagCM9 Qdot 655 multimers, were here combined with laser capture microdissection to detect and collect SIV Gag CM9 specific CD8+ T cells in lymph node tissue from SIV infected rhesus macaques. CD8+ T cells from SIV infected and uninfected rhesus macaques were also collected and compared to the SIV GagCM9 specific CD8+ T cells. Illumina bead array and transcriptional analyses were used to assess the transcriptional profiles and the three different CD8+ T cell populations displayed unique transcriptional patterns. This pilot study demonstrates that rapid and specific immunostaining combined with laser capture microdissection in concert with transcriptional profiling may be used to elucidate phenotypic differences between CD8+ T cells in SIV infection. Such technologies may be useful to determine differences in functional activities of HIV/SIV specific T cells.

Depot Medroxyprogesterone Acetate Use Is Associated With Elevated Innate Immune Effector Molecules in Cervicovaginal Secretions of HIV-1-Uninfected Women.

OBJECTIVE: The effects of sex hormones on the immune defenses of the female genital mucosa and its susceptibility to infections are poorly understood. The injectable hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may increase the risk for HIV-1 acquisition. We assessed the local concentration in the female genital mucosa of cationic polypeptides with reported antiviral activity in relation to DMPA use. METHODS: HIV-1-uninfected women were recruited from among couples testing for HIV in Nairobi, Kenya. Cervicovaginal secretion samples were collected, and the concentrations of HNP1-3, LL-37, lactoferrin, HBD-2, and SLPI were measured by enzyme-linked immunosorbent assays. Levels of cationic polypeptides in cervicovaginal secretions were compared between women who were not using hormonal contraception and those using DMPA, oral, or implantable contraception. RESULTS: Among 228 women, 165 (72%) reported not using hormonal contraception at enrollment, 41 (18%) used DMPA, 16 (7%) used an oral contraceptive, and 6 (3%) used a contraceptive implant. Compared with nonusers of hormonal contraception, DMPA users had significantly higher mean levels of HNP1-3 (2.38 vs. 2.04 log10 ng/mL; P = 0.024), LL-37 (0.81 vs. 0.40 log10 ng/mL; P = 0.027), and lactoferrin (3.03 vs. 2.60 log10 ng/mL; P = 0.002), whereas SLPI and HBD-2 were similar. CONCLUSIONS: Although all analyzed cationic polypeptides have intrinsic antiviral capacity, their interaction and cumulative effect on female genital mucosa susceptibility to infections in vivo has yet to be unraveled. This study suggests a potential mechanism underlying the effect of DMPA on the innate immune defenses, providing a rationale to investigate its effect on HIV-1 acquisition risk.

Progesterone-based intrauterine device use is associated with a thinner apical layer of the human ectocervical epithelium and a lower ZO-1 mRNA expression.

Currently, whether hormonal contraceptives affect male to female human immunodeficiency virus (HIV) transmission is being debated. In this study, we investigated whether the use of progesterone-based intrauterine devices (pIUDs) is associated with a thinning effect on the ectocervical squamous epithelium, down-regulation of epithelial junction proteins, and/or alteration of HIV target cell distribution in the human ectocervix. Ectocervical tissue biopsies from healthy premenopausal volunteers using pIUDs were collected and compared to biopsies obtained from two control groups, namely women using combined oral contraceptives (COCs) or who do not use hormonal contraceptives. In situ staining and image analysis were used to measure epithelial thickness and the presence of HIV receptors in tissue biopsies. Messenger RNA levels of epithelial junction markers were measured by quantitative PCR. The epithelial thickness displayed by women in the pIUD group was similar to those in the COC group, but significantly thinner as compared to women in the no hormonal contraceptive group. The thinner epithelial layer of the pIUD group was specific to the apical layer of the ectocervix. Furthermore, the pIUD group expressed significantly lower levels of the tight junction marker ZO-1 within the epithelium as compared to the COC group. Similar expression levels of HIV receptors and coreceptors CD4, CCR5, DC-SIGN, and Langerin were observed in the three study groups. Thus, women using pIUD displayed a thinner apical layer of the ectocervical epithelium and reduced ZO-1 expression as compared to control groups. These data suggest that pIUD use may weaken the ectocervical epithelial barrier against invading pathogens, including HIV.

Expression pattern of the PRDX2, RAB1A, RAB1B, RAB5A and RAB25 genes in normal and cancer cervical tissues.

Cervical cancer is the second most prevalent malignancy among women worldwide, and additional objective diagnostic markers for this disease are needed. Given the link between cancer development and alternative splicing, we aimed to analyze the splicing patterns of the PRDX2, RAB1A, RAB1B, RAB5A and RAB25 genes, which are associated with different cancers, in normal cervical tissue, preinvasive cervical lesions and invasive cervical tumors, to identify new objective diagnostic markers. Biopsies of normal cervical tissue, preinvasive cervical lesions and invasive cervical tumors, were subjected to rapid amplification of cDNA 3' ends (3' RACE) RT­PCR. Resulting PCR products were analyzed on agarose gels, gel­purified and sequenced. Normal cervical tissue, preinvasive cervical lesions and invasive cervical tumors contained one PCR product corresponding to full­length PRDX2, RAB5A and RAB25 transcripts. All tissues contained two RAB1A­specific PCR products corresponding to the full­length transcript and one new alternatively spliced RAB1A transcript. Invasive cervical tumors contained one PCR product corresponding to the full­length RAB1B transcript, while all normal cervical tissue and preinvasive cervical lesions contained both the full­length RAB1B transcript and three new alternatively spliced RAB1B transcripts. Alternative splicing of the RAB1A transcript occurs in all cervical tissues, while alternative splicing of the RAB1B transcript occurs in normal cervical tissue and in preinvasive cervical lesions; not in invasive cervical tumors.

A comparative proteomic analysis of the soluble immune factor environment of rectal and oral mucosa.

OBJECTIVE: Sexual transmission of HIV occurs across a mucosal surface, which contains many soluble immune factors important for HIV immunity. Although the composition of mucosal fluids in the vaginal and oral compartments has been studied extensively, the knowledge of the expression of these factors in the rectal mucosa has been understudied and is very limited. This has particular relevance given that the highest rates of HIV acquisition occur via the rectal tract. To further our understanding of rectal mucosa, this study uses a proteomics approach to characterize immune factor components of rectal fluid, using saliva as a comparison, and evaluates its antiviral activity against HIV. METHODS: Paired salivary fluid (n = 10) and rectal lavage fluid (n = 10) samples were collected from healthy, HIV seronegative individuals. Samples were analyzed by label-free tandem mass spectrometry to comprehensively identify and quantify mucosal immune protein abundance differences between saliva and rectal fluids. The HIV inhibitory capacity of these fluids was further assessed using a TZM-bl reporter cell line. RESULTS: Of the 315 proteins identified in rectal lavage fluid, 72 had known immune functions, many of which have described anti-HIV activity, including cathelicidin, serpins, cystatins and antileukoproteinase. The majority of immune factors were similarly expressed between fluids, with only 21 differentially abundant (p<0.05, multiple comparison corrected). Notably, rectal mucosa had a high abundance of mucosal immunoglobulins and antiproteases relative to saliva, Rectal lavage limited HIV infection by 40-50% in vitro (p<0.05), which is lower than the potent anti-HIV effect of oral mucosal fluid (70-80% inhibition, p<0.005). CONCLUSIONS: This study reveals that rectal mucosa contains many innate immune factors important for host immunity to HIV and can limit viral replication in vitro. This indicates an important role for this fluid as the first line of defense against HIV.

Bacteremia in Swedish hematological patients with febrile neutropenia: bacterial spectrum and antimicrobial resistance patterns.

BACKGROUND: The etiology of bacteremia in hematological patients with febrile neutropenia differs geographically and changes over time. Since efficient empirical antibiotic treatment depends on relevant knowledge of the bacterial panorama, the aim of this study was to describe the prevalence of bacteremia, the bacterial spectrum, and the resistance patterns of the isolates in this group today. METHODS: In a cross-sectional study, routine blood cultures from febrile episodes occurring in adult patients with hematological disorders and neutropenia presenting to Karolinska University Hospital, Stockholm, Sweden during a 24-month period, were analyzed. RESULTS: A total of 142 febrile neutropenic episodes occurring in 124 hematological patients were included in the study. Bacteremia was documented in 27% of the episodes, and of these, 58% were due to Gram-positive pathogens. The most common isolates were viridans streptococci, coagulase-negative staphylococci, and Escherichia coli. Low levels of antibiotic resistance were detected. The underlying diagnosis of non-Hodgkin's lymphoma (NHL) was independently negatively associated with documented bacteremia (p < 0.01). CONCLUSIONS: The prevalence of bacteremia and the bacterial spectrum were consistent with recent Scandinavian reports. Substantially lower levels of antimicrobial resistance were registered compared to those found in other European centers. Patients with NHL were less likely to have documented bacteremia in this study.