Matters of life and death: How estrogen and estrogen receptor binding to the immunoglobulin heavy chain locus may influence outcomes of infection, allergy, and autoimmune disease.

Sex hormones are best known for their influences on reproduction, but they also have profound influences on the immune response. Examples of sex-specific differences include: (i) the relatively poor control of influenza virus infections in males compared to females, (ii) allergic asthma, an IgE-associated hypersensitivity reaction that is exacerbated in adolescent females compared to males, and (iii) systemic lupus erythematosus, a life-threatening autoimmune disease with a 9:1 female:male bias. Here we consider how estrogen and estrogen receptor α (ERα) may influence the immune response by modifying class switch recombination (CSR) and immunoglobulin expression patterns. We focus on ERα binding to enhancers (Eµ and the 3' regulatory region) and switch sites (Sµ and Sε) in the immunoglobulin heavy chain locus. Our preliminary data from ChIP-seq analyses of purified, activated B cells show estrogen-mediated changes in the positioning of ERα binding within and near Sµ and Sε. In the presence of estrogen, ERα is bound not only to estrogen response elements (ERE), but also to adenosine-cytidine (AC)-repeats and poly adenosine (poly A) sequences, in some cases within constant region gene introns. We propose that by binding these sites, estrogen and ERα directly participate in the DNA loop formation required for CSR. We further suggest that estrogen regulates immunoglobulin expression patterns and can thereby influence life-and-death outcomes of infection, hypersensitivity, and autoimmune disease.

Retinol binding protein and vitamin D associations with serum antibody isotypes, serum influenza virus-specific neutralizing activities and airway cytokine profiles.

Vitamin A supports the induction of immunoglobulin (Ig)A responses at mucosal surfaces in mice, but much less is known about the influence of vitamins on antibody isotype expression in humans. To address this knowledge gap, we examined 46 residual blood samples from adults and children, some of whom were experiencing influenza virus infections of the respiratory tract. Assays were performed for retinol binding protein (RBP, a surrogate for vitamin A), vitamin D (a related vitamin) and antibody isotypes. Results showed that all but two tested samples exhibited RBP and/or vitamin D insufficiencies or deficiencies. Vitamin D correlated with blood IgM and IgG3, while RBP correlated with IgG4 and IgA. RBP also correlated positively with age and with influenza virus-specific antibody neutralization titres. Individuals with low blood RBP levels exhibited the highest frequencies of over-expressed cytokines and growth factors in nasal wash samples, an indication of inflamed mucosal tissues. While cause-effect relationships were not discerned, results support a hypothesis that vitamins directly influence B cell isotype expression in humans, and by so doing may help protect mucosal surfaces from respiratory viral disease.

Squamous cell carcinoma arising in a dermoid cyst of the ovary: a case series.

OBJECTIVE: Malignant transformation in a dermoid cyst of the ovary is a rare complication, occurring in only 1-2% of cases, with squamous cell carcinoma being the most common type. Preoperative diagnosis is difficult because of the lack of specific symptoms and signs to suggest malignancy. Because of the small numbers of women involved, our knowledge of this rare tumour type is limited. This study aims to further characterise the population of women affected, the disease itself and the most appropriate management strategy. DESIGN: We identified 14 women with this diagnosis between 1989 and 2006. This is a descriptive study, looking at the patient characteristics, mode of presentation and the role of tumour markers and radiological imaging in diagnosis. We also examined the stage and pathological features of the tumour at presentation and the subsequent course of the disease. We have also described our experiences using surgery, chemotherapy and radiotherapy in the management of these women. RESULTS: We found that these tumours present at an age older than that of mature teratomas and that there are no reliable diagnostic tools or prognostic indicators. The behaviour of these tumours is unpredictable, and the role of chemotherapy and radiotherapy remains unclear. We suggest that repeated surgical resection of disease at the time of relapse could give a very durable response in selected women.

Individual HIV type 1 envelope-specific T cell responses and epitopes do not segregate by virus subtype.

HIV-1 vaccines are often designed to target one or several virus subtype(s). They therefore include antigens (e.g., env or env/gag/pol) from each targeted subtype to elicit subtype-directed immunity. To determine if individual T cells respond to HIV-1 antigens in a subtype-directed manner, we selected four T cell hybridomas, each representative of a different immunodominant response toward a subtype B envelope. Hybridomas were tested for responses toward 20 subtype B envelope proteins and one protein each from subtypes A, C, and D. None of the hybridomas cross-reacted with all subtype B envelopes, yet three responded to a non-B protein. Core epitopes and flanking regions affected responsiveness. This lack of subtype-directed activity was corroborated by analyses of the Los Alamos database; like immune responses, epitope distributions were not dictated by subtype. Results highlight the difficulty of predicting immune responses based on subtype alone and encourage considerations of antigenic disparity in addition to subtype disparity during HIV-1 vaccine design.

Vitamin Supplementation at the Time of Immunization with a Cold-Adapted Influenza Virus Vaccine Corrects Poor Mucosal Antibody Responses in Mice Deficient for Vitamins A and D.

Vitamin A and D deficiencies and insufficiencies are prevalent worldwide in developed and developing countries. Vitamin metabolites are functionally intertwined in that they are high-affinity ligands for related receptors of the nuclear receptor superfamily. The effects of vitamin A deficiencies (VAD) on antibody responses to respiratory virus vaccines have already been demonstrated. Of particular concern was the reduction in IgA, a first line of defense against pathogens in the respiratory tract. Here, we describe the individual and combined effects of vitamin A and D deficiencies in mice immunized with an attenuated influenza virus vaccine. Relative to VAD, vitamin D deficiency (VDD) had a limited effect, but double deficiencies for vitamins A and D (VAD+VDD) further reduced antibody responses in the respiratory tract. The administration of supplemental vitamins A and D to VAD+VDD mice at the time of vaccination restored responses in a dose-dependent manner. Results suggest that vitamin supplementation programs may be beneficial in a clinical setting to promote healthy immune responses to respiratory virus vaccines in vitamin-deficient individuals.

Importance of abortive shock capability with electrogram storage in cardioverter-defibrillator devices.

OBJECTIVES: This study evaluates the ability of a third-generation cardioverter-defibrillator to abort energy delivery and the importance of electrogram storage in analyzing the aborted events. BACKGROUND: In the Cadence Tiered Therapy Defibrillator, when a tachycardia satisfies detection criteria for cardioversion or defibrillation therapy, high voltage capacitors begin charging. The Cadence defibrillator continues monitoring the rhythm during charging and if the rate decreases to below the rate triggering therapy, charging is terminated. This event is registered as an aborted shock. The defibrillator also has the ability to store intracardiac electrogram recordings of the electrical events that precipitate device therapy or aborted shocks. METHODS: During a mean follow-up interval of 10 +/- 7 months, 55 aborted events were registered by the Cadence defibrillator in 18 of the 49 patients who received it. Thirty-two stored ventricular electrograms of events leading to aborted shocks were available for analysis in 15 patients. RESULTS: Intracardiac electrogram recordings demonstrated the probable electrical events leading to these aborted shocks included nonsustained ventricular tachycardia (n = 10), nonsustained rapid polymorphic ventricular tachycardia/ventricular fibrillation (n = 2), atrial fibrillation (n = 5), supraventricular tachycardia (n = 2) and electrical noise (n = 13). Eleven patients had a therapeutic intervention initiated as a consequence of the diagnostic information provided by analysis of intracardiac electrogram recordings. Four of the 15 patients had no changes made. During a follow-up period of 9 +/- 5 months after therapy was altered, no patient had subsequent aborted shocks. Five patients have had seven appropriate shocks for sustained ventricular tachycardias. CONCLUSIONS: The ability of Cadence defibrillator to continue tachycardia sensing during capacitor charging and to abort shock therapy for self-terminating events prevented unnecessary shocks in 18 (37%) of the 49 patients. Intracardiac electrogram recordings were critical for instituting appropriate therapy that may have prevented unnecessary device charging and inappropriate discharges.

Profound abnormality of the B/T lymphocyte ratio during chemotherapy for pediatric acute lymphoblastic leukemia.

The fluorescence-activated cell sorter (FACS) was utilized to phenotype lymphocyte compartments in children receiving intensive chemotherapy for acute lymphoblastic leukemia (ALL). Sixteen patients (eight males and eight females) of diverse ages, risks of relapse, and within weeks 7-53 of maintenance/continuation chemotherapy treatment were arbitrarily selected for study. All 16 patients had profound B cell lymphopenia. In contrast, T cell numbers were often normal or marginally low, and accounted for up to 98% of the lymphocyte populations. No abnormality in T cell phenotypes could be demonstrated. Due to the highly skewed B/T lymphocyte ratios in these ALL patients, the absolute white blood cell counts and lymphocyte percentages were not predictive of the underlying B cell lymphopenia. Patients were also tested for serum immunoglobulin levels and most had abnormally low IgG and IgM. None of four patients immunized with the 1996-1997 influenza virus vaccine seroconverted to at least two vaccine antigens as compared to 10 of 10 healthy, age-matched controls. In total, these data highlight for the first time the profound abnormality of the B/T lymphocyte ratio in patients during treatment for ALL, and argue for consideration of B cell-targeted immunotherapy.

Gene rearrangement patterning and DNase-I hypersensitive sites within the T-cell receptor J alpha locus.

For several years, the relationship between alpha beta and gamma delta T-cell progenitors has been a topic of debate. Some argue that a subset of T-cell progenitors is "pre-committed" to the alpha beta lineage and is thus programmed to rearrange alpha, but not delta genes. It is further argued that the deletion of the delta locus by a unique rearrangement, delta rec-psi J alpha, may be the critical forerunner to V-J alpha joins in alpha beta committed cells and that a hypersensitive site (HS) termed 5'TEA might regulate such rearrangement. Here we present an alternative hypothesis. We first emphasize that directed J alpha gene rearrangements do not exclusively target the psi J alpha gene, but that clustered gene rearrangements occur throughout the J alpha locus during T-cell development. We describe the existence of not one, but at least two HS sites distributed along the J alpha locus which might serve as regulators for the gene rearrangement event. Finally, we suggest that progenitor T-cells are not committed to a particular delta or alpha gene rearrangement, but that a flexible progenitor responds to complex interactions between environmental signals and multiple regulatory elements interspersed among delta/alpha genes.

Anatomical aspects of the superior olivary complex.

The cytology of the superior olivary complex was studied in nissl stained sections of eight human brainstems, including adult, infant and fetus, and in the brains of ten juvenile rhesus monkeys. The most prominent components of the superior olivary complex of primates were specifically investigated, i.e. the medial (SOM) and lateral (SOL) superior olivary nuclei. Cell counts of these segments were done in human brainstems. The adult SOM was comprised of an average of 11,428 (7,850--15,010) perikarya; the SOL contained an average of 3,923 (2,890--5,400) neurons. These findings indicate that the SOL contains as many cells as reported in other primates, and is not reduced. The SOL appears somewhat inconspicuous in the human because it is organized into as many as six clusters of cells rather than forming one well circumscribed configuration as in the monkey and cat. The total cell population of the SOM together with the SOL was approximately the same on each side of individual brains. If one segment was larger on one side than the opposite side, the other segment was correspondingly reduced to maintain the relative symmetry. This suggests that a single mechanism controls the cell complement of at least two segments of the superior olivary complex.

T cell immunotherapeutic populations control viral infections in bone marrow transplant recipients.

Immunotherapies designed to prevent infection serve as an increasingly important adjunct to bone marrow transplantation (BMT). T cell immunotherapies are particularly useful for the control of virus infections, provided that T cell populations are free of graft-vs-host (GVH) activity. In this review, we describe positive and negative selection methods with which donor T cell populations devoid of GVH activity can be prepared for transfer to the immunodeficient BMT recipient. The support of patients with T cell immunotherapies may ultimately revolutionize BMT, elevating the procedure from a salvage to a front-line treatment strategy for otherwise fatal disorders.

Multi-envelope HIV vaccine safety and immunogenicity in small animals and chimpanzees.

A significant obstacle to HIV vaccine development lies in the remarkable diversity of envelope proteins, the major targets of neutralizing antibody. That envelope diversity must be targeted is demonstrated by results from nonhuman primate studies in which single-envelope vaccines have protected against homologous, but rarely against heterologous virus challenges. Similarly, in clinical trials, single-envelope vaccines have failed to prevent break-through infections when challenge viruses were inevitably mismatched with the vaccine. To protect humans from infection by any isolate of HIV, we have prepared vaccine cocktails combining multiple envelopes from distinct viral isolates. We have tested several vehicles for vaccine delivery in small animals and have shown that successive immunizations with envelope, presented first as a DNA recombinant, then as a vaccinia virus (VV) recombinant, and finally as purified protein elicited strong neutralizing antibody responses. We have also tested the VV recombinant vaccine in chimpanzees. Pairs of animals received either single- or multi-envelope VV recombinant vaccines administered by the subcutaneous route. Results showed that the multi-envelope vaccine was safe, immunogenic, and superior to the single-envelope vaccine in eliciting HIV-specific antibody measurable in a standard clinical, immune assay. The promise of this system has led to the initiation of clinical trials, with which the hypothesis that cocktail vaccines will prevent human HIV infections may ultimately be tested.

Restricted usage of T-cell receptor V alpha sequence and variable-joining pairs after normal T-cell development and bone marrow transplantation.

TCR V alpha 3 and V alpha 5 transcripts in PBLs from healthy individuals of multiple age groups and from BMT recipients were analyzed. PCR, cloning, and sequencing studies revealed significant V-J junctional diversity among TCR transcripts from all tested blood samples, as provided both by N/P-region addition and exonuclease activity. However, results illustrated restrictions in TCR alpha diversity at several additional levels. First, V alpha 5 and V alpha 3 gene families, which were expected to be composed of multiple members, were dominated in each case by a single sequence at the transcript level. Second, restrictions existed in V-J pairing in that J alpha genes, which were encoded toward the 5' region of the locus, were rearranged frequently with V alpha 3 and rarely with V alpha 5. Conversely, J alpha genes encoded toward the 3' region of the locus preferentially rearranged with V alpha 5. Healthy individuals showed few differences with regard to V-J pairing patterns, while one of three BMT recipients demonstrated a skewed usage of 3' J alpha genes. In total, results demonstrated qualitative restrictions that may limit the working TCR repertoire in human peripheral tissues, both among BMT recipients and their healthy donors.

Structural features of HIV envelope defined by antibody escape mutant analysis.

Two neutralizing antibodies specific for the V3 sequence of HIV envelope were used to generate escape variants from the HTLV(IIIB) founder virus. The full gp120 sequence of each variant was then analyzed to identify mutations responsible for immune escape. As predicted, most escape variants harbored amino acid changes in the V3 crown sequence. However, one variant differed from its founder only within the conserved C2 region. These findings, when analyzed in conjunction with crystallographic data, suggest a new three-dimensional model for HIV envelope folding, in which the V3 loop extends across the CD4-binding face of gp120 to associate with discontinuous C2 residues. This envelope configuration may provide an effective immune defense mechanism for HIV, as the highly variable residues of the V3 loop may shield conserved amino acids pertinent to viral infection.

Antibody and pre- plus post-transplant prednisone treatments support T cell-depleted stem cell engraftment without drug-induced morbidity.

Rigorous T cell depletion methods can now be used to reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic, hematopoietic stem cell transplantation (HSCT). However, full T cell depletion is also associated with a significant risk of graft failure. Here we hypothesize that engraftment failures after T cell-depleted HSCT may be due, in part, to the absence of GVHD prophylaxis. To test this hypothesis, we used a haploidentical mouse model to systematically measure the effects of immunosuppressive drug treatments and anti-T cell antibodies on engraftment. Results showed that engraftment was supported in all animals when hosts were pre-treated with anti-T cell antibodies, but donor chimerism was significantly improved when hosts were also treated with prednisone. Interestingly, when hosts received only pre-HSCT prednisone treatments, engraftment was not improved; when hosts received only post-HSCT prednisone (initiated near the time of irradiation), the animals became extremely ill. Results therefore demonstrated the need for both pre- and post-HSCT prednisone treatments as a means to ensure engraftment without morbidity in all host animals.

Eliminating graft-versus-host potential from T cell immunotherapeutic populations.

It is common knowledge that donor T cells are responsible for graft-versus-host disease (GVHD) following bone marrow transplantation (BMT), yet GVHD remains a grave threat to transplant patients. The donor marrow can be purged of T cells to reduce this danger, but the risks of viral infections, tumor relapse and graft rejection are then increased. Here we describe a method that may be used to provide BMT patients with T cell immunotherapeutic populations responsive to foreign antigens, but unresponsive to host HLA. The method involves the culture of donor T cells with host-derived B lymphoblastoid cell lines (BLCL). During culture, the T cells are activated by the mismatched host HLA. Activated cells are subsequently removed by fluorescence-activated cell sorting. Criteria for removal include cell size and the expression of multiple T cell activation antigens on cell membranes. After the procedure, T cell populations retain helper and cytotoxic T cell responses against foreign antigens, but are specifically devoid of responses to host HLA. This technique offers a promising method for providing BMT patients the benefits of T cell immunity without the consequences of GVHD.

Reconstitution of the T cell receptor alpha beta repertoire in recipients of allogeneic BMT.

This report describes the qualitative analysis of T cell receptor (TCR) repertoire regeneration in recipients of BMT. RNA samples from patient and control peripheral blood lymphocytes were prepared and tested for the presence of multiple V alpha and V beta transcripts by the polymerase chain reaction. TCR V gene expression was highly diverse within the first 6 months post-transplantation in recipients receiving either T cell-depleted or T cell-replete marrow, and in HLA mismatched as well as matched donor-recipient pairs. The sequencing of TCR message from BMT recipients also demonstrated J gene diversity and apparently normal junctional diversity at the V-J alpha join. Thus, T cell pools in BMT recipients are largely heterogeneous, not mono- or oligoclonal.

Effect of extended immunosuppressive drug treatment on B cell vs T cell reconstitution in pediatric bone marrow transplant recipients.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for a variety of malignancies and blood disorders, but rarely serves as a frontline treatment because of numerous, potential complications. Important and frequent complications relate to the profound immunosuppression that inevitably occurs during the first several months following treatment. To better elucidate and subsequently improve immune reconstitution, we examined T and B cell subsets among 43 pediatric BMT recipients in a retrospective study. We found that the relative numbers of T cells and B cells (T:B ratios) were discordant and highly variable among patients at day approximately 100 after BMT. Further investigation of BMT parameters identified a strong correlation between T:B ratios and immunosuppressive drug treatments, providing an explanation for variable lymphocyte reconstitution profiles. Results suggest that: (1) immunosuppressive therapy inhibits B cell expansion more strongly than T cell expansion following BMT; (2) WBC and absolute lymphocyte counts fail to reveal profound B cell immunodeficiencies in some BMT patients; and (3) routine analyses of T:B ratios serve to identify patients warranting close follow-up and extended supportive immunotherapy.

Oral retinyl palmitate or retinoic acid corrects mucosal IgA responses toward an intranasal influenza virus vaccine in vitamin A deficient mice.

Vitamin A deficiency (VAD) is a leading cause of pediatric morbidity and mortality due to infectious diseases. Recent pre-clinical studies have revealed that VAD impairs mucosal IgA-producing antibody forming cell (AFC) responses toward a paramyxovirus vaccine in the upper respiratory tract (URT), thus impeding a first line of defense at the pathogen's point-of-entry. The studies described here tested the hypothesis that VAD may also impair immune responses after FluMist vaccinations. Results show that (i) IgA-producing antibody forming cells (AFCs) are significantly reduced following FluMist vaccination in VAD mice, and (ii) oral doses of either retinyl palmitate or retinoic acid administered on days 0, 3, and 7 relative to vaccination rescue the response. Data encourage the conduct of clinical studies to determine if there are FluMist vaccine weaknesses in human VAD populations and to test corrective supplementation strategies. Improvements in vaccine efficacy may ultimately reduce the morbidity and mortality caused by influenza virus worldwide.

Inhibition of primary clinical isolates of human parainfluenza virus by DAS181 in cell culture and in a cotton rat model.

DAS181 is a novel drug in development for the treatment of influenza as well as human parainfluenza viruses (hPIVs). Previous studies demonstrated that DAS181 inhibited laboratory strains of hPIV, but no tests were conducted with primary clinical isolates of hPIV. To fill this gap, we studied six primary isolates including hPIV-2 and hPIV-3. First tests showed that the amplification of all viruses in vitro was reproducibly inhibited with DAS181 drug concentrations ranging between 0.1 and 1nM. An hPIV-3 primary clinical isolate was then tested in a cotton rat model for sensitivity to 0.3-1mg/kg drug treatments. Results showed that virus amplification in the lower respiratory tract was significantly and reproducibly inhibited by drug. Together, experiments demonstrated that DAS181 inhibited primary clinical isolates of hPIV in vitro and in vivo at doses similar to those previously described for inhibition of laboratory hPIV and influenza virus isolates.