Oral health-related quality of life before hematopoietic stem cell transplantation.

OBJECTIVES: This study was planned to evaluate the impact of oral health on the quality of life (QoL) of patients undergoing hematopoietic stem cell transplantation (HSCT). MATERIAL AND METHODS: We assessed 200 patients divided into two paired groups: 100 patients prior to HSCT (SG) and 100 healthy volunteers (CG). We applied the Oral Health Impact Profile instrument, which is based on the biopsychosocial problem gradation of World Health Organization (WHO) and relates oral health problems with QoL according to seven dimensions. RESULTS: Fourteen patients in SG were referred for extraction of one to eight teeth, mostly due to deep caries with risk of pulpal infection and possible spread of infection via blood (r = 0.59, p = 0.000). The presence of severely compromised teeth by extensive decay correlated with Oral Health Impact Profile (OHIP-14). The Mann-Whitney test showed a significant difference between SG and CG in the following dimensions: functional limitation (p < 0.001), physical pain (p = 0.025), physical disability (p = 0.016), and social disability (p = 0.01). CONCLUSIONS: The impact of oral health on QoL of onco-hematologic patients is weak but is greater as compared to healthy ones. Nevertheless, a significant impact is seen in patients with severely compromised teeth. CLINICAL RELEVANCE: The negligence of oral care, proper hygiene, and the search for dental care may increase the risk for local and systemic infections.

Impact of mother donor, peripheral blood stem cells and measurable residual disease on outcomes after haploidentical hematopoietic cell transplantation with post-transplant cyclophosphamide in children with acute leukaemia.

Haploidentical hematopoietic-cell transplantation using post-transplant cyclophosphamide(Haplo-PTCy) is a feasible procedure in children with haematologic malignancies. However, data of a large series of children with acute leukaemia(AL) in this setting is missing. We analysed 144 AL Haplo-PTCy paediatric recipients; median age was 10 years. Patients had acute lymphoblastic(ALL; n = 86) or myeloblastic leukaemia(AML; n = 58) and were transplanted in remission(CR1: n = 40; CR2: n = 57; CR3+: n = 27) or relapse (n = 20). Bone marrow was the graft source in 57%; donors were father (54%), mother (35%), or sibling (11%). Myeloablative conditioning was used in 87%. Median follow-up was 31 months. At day +100, cumulative incidence (CI) of neutrophil recovery and acute GVHD (II-IV) were 94% and 40%, respectively. At 2-years, CI of chronic GVHD and relapse, were 31%, 40%, and estimated 2-year overall survival (OS), leukaemia-free survival (LFS) and graft-versus-host-relapse-free survival (GRFS) were 52%, 44% and 34% respectively. For patients transplanted in remission, positive measurable residual disease (MRD) prior to transplant was associated with decreased LFS (p = 0.05) and GRFS (p = 0.003) and increased risk of relapse (p = 0.02). Mother donor was associated with increased risk of chronic GVHD (p = 0.001), decreased OS (p = 0.03) and GRFS (p = 0.004). Use of PBSC was associated with increased risk of chronic GVHD (p = 0.04). In conclusion, achieving MRD negativity pre-transplant, avoiding use of mother donors and PBSC as graft source may improve outcomes of Haplo-PTCy in children with AL.

Congenital Zika syndrome is associated with maternal protein malnutrition.

Zika virus (ZIKV) infection during pregnancy is associated with a spectrum of developmental impairments known as congenital Zika syndrome (CZS). The prevalence of this syndrome varies across ZIKV endemic regions, suggesting that its occurrence could depend on cofactors. Here, we evaluate the relevance of protein malnutrition for the emergence of CZS. Epidemiological data from the ZIKV outbreak in the Americas suggest a relationship between undernutrition and cases of microcephaly. To experimentally examine this relationship, we use immunocompetent pregnant mice, which were subjected to protein malnutrition and infected with a Brazilian ZIKV strain. We found that the combination of protein restriction and ZIKV infection leads to severe alterations of placental structure and embryonic body growth, with offspring displaying a reduction in neurogenesis and postnatal brain size. RNA-seq analysis reveals gene expression deregulation required for brain development in infected low-protein progeny. These results suggest that maternal protein malnutrition increases susceptibility to CZS.

Guidelines on good clinical laboratory practice: bridging operations between research and clinical research laboratories.

A set of Good Clinical Laboratory Practice (GCLP) standards that embraces both the research and clinical aspects of GLP were developed utilizing a variety of collected regulatory and guidance material. We describe eleven core elements that constitute the GCLP standards with the objective of filling a gap for laboratory guidance, based on IND sponsor requirements, for conducting laboratory testing using specimens from human clinical trials. These GCLP standards provide guidance on implementing GLP requirements that are critical for laboratory operations, such as performance of protocol-mandated safety assays, peripheral blood mononuclear cell processing and immunological or endpoint assays from biological interventions on IND-registered clinical trials. The expectation is that compliance with the GCLP standards, monitored annually by external audits, will allow research and development laboratories to maintain data integrity and to provide immunogenicity, safety, and product efficacy data that is repeatable, reliable, auditable and that can be easily reconstructed in a research setting.

Individual variation is the key to the development of a vaccine against Staphylococcus aureus: a comparative study between mice lineages.

Bacterial infections occur worldwide and are a major public health problem. Among pathogens, Staphylococcus aureus is the main causative agent of bacterial diseases in the world. This study aimed to evaluate which components of the immune system could act protectively against a S. aureus infection in intradermally immunized mice. C57BL/6 and A/j mice were immunized intradermally with S. aureus inactivated by heat and then challenged with viable strains in an air pouch model. At 6, 12, and 24 h after the challenge, euthanasia was performed, and the cellular profile of the inflammatory infiltrate, cytokines, and the bacterial load were evaluated in the air pouch lavages. Immunized mice demonstrated that the intradermal immunization with S. aureus promoted protection in C57BL/6 mice by reducing the bacterial, which was correlated with increased serum concentration of IgG antibodies (IgG1 and IgG2a) against S. aureus. The increase in IgG2a antibody levels was correlated with a decrease of bacterial load in intradermally immunized C57BL/6 mice, along with production of IL-17A at the inflammation site, as well as IgG1consumption. Similar results were not found in the A/j lineage. In conclusion, a vaccine against S. aureus should focus more on the individual characteristics of the host because it is a determinant factor for the success of the immunization.

Six cases of leprosy associated with allogeneic hematopoietic SCT.

We report here the first six cases of leprosy associated with HLA-identical allogeneic SCT in different phases and with different findings and outcomes. Skin and peripheral nerves may be sites of leprosy associated with SCT, stressing the importance of differential diagnosis between leprosy and GVHD or drug reactions. Clinical manifestations of leprosy before or after transplantation did not influence the outcome of SCT in our cases.

Adenine nucleotide efflux in mitochondria induced by inorganic pyrophosphate.

The efflux of mitochondrial adenine nucleotide which is induced by addition of PPi to suspensions of rat liver mitochondria has been investigated. This efflux of adenine nucleotide is greatly stimulated by the uncoupler FCCP at 1 microM, Vmax being 6.7 nmol/min per mg protein as compared to 2.0 nmol/min per mg protein in its absence. The depletion process is inhibited by carboxyatractyloside. The Km for PPi of 1.25 mM is essentially unchanged when uncoupler is added. Quantitation of the individual adenine nucleotide species (ATP, ADP and AMP) and their relationship to the rate of efflux suggests that ADP is the predominant species being exchanged for PPi.

Isolation of a sealed homogeneous population of inner membrane fragments with inverted orientation from rat liver mitochondria using specific lectin immunoprecipitation.

A novel method for the isolation of well-defined populations of inside-out vesicles from rat liver mitochondria is described. The technique utilizes specific immunoprecipitation of vesicles with accessible carbohydrate residues from a mixed population of inner membrane fragments using wheat germ agglutinin and anti-wheat germ agglutinin IgG. The unprecipitated fraction comprises 30--50% of the original population and exhibits little or no cytochrome c oxidase activity as estimated with exogenous cytochrome c as substrate. Addition of deoxycholate to promote membrane disruption results in an 8--10-fold increase in enzymic activity compared to only 1.5--2.0-fold stimulation in standard preparations of submitochondrial particles. It is concluded that the lectin affinity-purified membranes represent a sealed homogeneous (90--95% pure) population of inside-out inner membrane vesicles.

Lectins as biochemical agents for the isolation of sealed membrane vesicles of defined polarity.

The asymmetric distribution of carbohydrate on biological membranes has provided the basis for the development of lectin-affinity methodology which permits the isolation of sealed, inside-out membrane fractions from heterogeneous populations of vesicles. Optimal conditions for these separations have been assessed employing purified right-side-out and inside-out vesicles derived from the plasma membrane of human erythrocytes as a model system. In this special case, homogeneous populations of defined polarity can be produced by varying the ionic conditions during formation of the vesicles. Surface-specific enzymic markers exist also for monitoring the integrity and orientation of a given population. Multivalent lectins such as wheat germ agglutinin and soya bean agglutinin which induce direct agglutination of erythrocyte membrane fragments containing accessible carbohydrate residues, selectively remove more than 90% of right-side-out and non-sealed membrane from a mixed population, a reaction which is inhibited by GluNAc or GalNAc, respectively. Non-agglutinating lectins, e.g. concanavalin A, immobilized on an inert matrix such as Sepharose 4B, may be employed to adsorb out specifically vesicles with exposed glycopeptides on their surface. In this technique, it is necessary normally to remove the non-sealed membranes on Dextran density gradients prior to the final preparation of inside-out vesicles on Con A-Sepharose. Finally, selective immunoprecipitation of fragments with accessible sugars may also be achieved after treatment with a non-agglutinating lectin (concanavalin A) followed by incubation with anti-concanavalin A IgG which promotes rapid aggregation of membrane containing exposed receptors for the lectin. These procedures should prove generally suitable for the isolation of tightly-sealed, inside-out membrane populations in a variety of biological systems. Pure populations of vesicles, exhibiting reversed polarity, are valuable in surface-labelling studies for investigating the structure, function and transmembrane distribution of integral membrane proteins/glycoproteins.

Haploidentical BMT and post-transplant Cy for severe aplastic anemia: a multicenter retrospective study.

Patients with refractory severe aplastic anemia (SAA) who lack a matched sibling or unrelated donor need new therapeutic approaches. Hematopoietic SCT (HSCT) using mismatched or haploidentical related donors has been used in the past, but was associated with a significant risk of GVHD and mortality. Recently, the use of post-transplant cyclophosphamide (Cy) has been shown to be an effective strategy to prevent GVHD in recipients of haploidentical HSCT, but the majority of reports have focused on patients with hematology malignancies. We describe the outcome of 16 patients who underwent haploidentical transplantation using a reduced-intensity conditioning regimen with post-transplant Cy. Stem cell sources were BM (N=13) or PBSCs (N=3). The rate of neutrophil engraftment was 94% and of platelet engraftment was 75%. Two patients had secondary graft failure and were successfully salvaged with another transplant. Three patients developed acute GVHD being grades 2-4 in two. Five patients have died and the 1-year OS was 67.1% (95% confidence interval: 36.5-86.4%). In our small series, the use of a reduced-intensity conditioning with post-transplant Cy in haploidentical BMT was associated with high rates of engraftment and low risk of GVHD in patients with relapsed/refractory SAA.

Evaluation of monoclonal antibodies to HIV-1 by neutralization and serological assays: an international collaboration. Collaborating Investigators.

In a National Institutes of Health (NIH)/World Health Organization (WHO)-sponsored collaboration, 26 laboratories characterized a coded panel of monoclonal antibodies (MAb) to HIV-1 envelope protein. The MAb were evaluated by serological [radioimmunoprecipitation, immunoblot, enzyme-linked immunosorbent assay (ELISA) and peptide mapping] and neutralization assays. Although laboratories used diverse neutralization assays that vary considerably in sensitivity, qualitatively similar data were obtained. The MAb were classified into three neutralization specificities: type-specific for MN and SF2, type-specific for IIIB, and group-specific for MN, SF2, and IIIB. The group-specific MAb displayed much lower neutralizing titers than the type-specific MAb. The specificity of MAb for neutralization was greater than for serological recognition of gp120 protein or peptide epitopes. Some MAb that bound to the same or closely overlapping linear epitopes had very different neutralization properties. The distinction between serological recognition and neutralization may result from differences in affinity of the MAb or may indicate that MAb can neutralize by interactions at a site distinct from the antibody binding site.

Evaluation of monoclonal antibodies to HIV-1 envelope by neutralization and binding assays: an international collaboration.

OBJECTIVE: To characterize a purified panel of monoclonal antibodies (MAb) to epitopes in HIV-1 envelope V3, CD4-binding region, C4 and gp41. DESIGN: Neutralization and/or binding activity data were obtained from 21 laboratories on a coded panel consisting of seven human MAb, seven mouse MAb, recombinant human CD4 immunoadhesin [CD4-immunoglobulin G (IgG)], normal human and normal murine Ig. METHODS: Laboratories performed a variety of neutralization assays and antigen binding assays with HIVIIIB, HIVMN and other laboratory strains of HIV-1. RESULTS: For a single MAb, there was up to a 10(3) range of neutralizing antibody titers between laboratories. The range in titers appeared to depend on the sensitivity of the neutralization assay. Two methods were used to consolidate the data from all laboratories, the geometric mean titer (GMT) and the median neutralizing titer (MNT). The panel of MAb were also analyzed by a variety of assays that measure binding activity to native or denatured epitopes. The relative binding activity of the MAb did not appear to correlate with neutralizing activity. CONCLUSION: Neutralization results from any single laboratory did not correlate with the collective data. The relative potency (rank order) of the MAb in the panel were equivalent when determined by GMT or MNT. These values may be useful to individual laboratories for estimating the sensitivity of their neutralization assays. The study also identified potential reference reagents with which neutralizing activity could be compared.

Neutralization of primary HIV-1 isolates by anti-envelope monoclonal antibodies.

OBJECTIVE: To evaluate human monoclonal antibodies (MAb) for neutralizing activity against primary HIV-1 isolates in peripheral blood mononuclear cells. DESIGN: Neutralization activity data were obtained from 11 laboratories on a coded panel consisting of six human MAb to HIV envelope V3, CD4-binding region or gp41. Hyperimmune globulin against HIV-1 and normal human immunoglobulin G were supplied as controls. Each laboratory received pre-titered virus for use in the studies. METHODS: Each laboratory measured neutralization of the MAb against laboratory strain HIVMN, genomic clone HIVJR-CSF, two subtype B and one subtype D primary isolates. RESULTS: The titers of the centrally supplied virus stocks as determined by re-titration or back-titration varied among laboratories and were generally 10-100-fold less than provided. The neutralizing activity of each MAb varied by as much as a 1000-fold among laboratories. These differences may result from varying sensitivity in neutralization assay protocols and the differing susceptibility of primary cells to infection with HIV-1. CONCLUSIONS: To consolidate the data from multiple laboratories, the neutralization titers were compared by classifying antibodies as neutralizing if the antibody concentration for 50% virus inhibition was < or = 10 micrograms/ml. By this criterion, the CD4-binding region and gp41 MAb neutralized all four subtype B viruses and the subtype D isolate in a few of the laboratories. The V3 MAb neutralized only HIVMN and the closely related HIVJR-CSF viruses.

Lysosomal membrane glycoproteins: properties of LAMP-1 and LAMP-2.

Several properties of the lysosomal membrane glycoproteins LAMP-1 and LAMP-2 have been analysed. Each molecule was strongly associated with lysosome membranes and was extracted only in the presence of detergent. Studies of the biosynthesis and processing of the glycoproteins showed that each contained a polypeptide core of approx. 43,000 Da as identified by use of tunicamycin and endoglycosidase H. Nascent glycoproteins pulse-labelled for 5 min with [35S]methionine were approx. 92,000 Da. These precursor molecules were processed in 30 min to highly heterogeneous mature glycoproteins of approx. 110,000 Da(LAMP-1) and 105,000 Da(LAMP-2). Concomitant with the increase in apparent Mr the molecules became endoglycosidase H resistant and acquired sialic acid residues, indicating that they were converted to complex-type oligosaccharides. The final maturation of the glycoproteins was blocked by monensin. Immunohistochemical analysis of tissues from Balb/c and Beige/J mice showed that the molecules were present on many types of cells, consistent with their presence in lysosomes. The patterns of tissue expression of LAMP-1 and LAMP-2 in the two mouse strains were the same except that the intensity of staining of LAMP-2 was less than that of LAMP-1. LAMP-2, but not LAMP-1, gave a decreased immunofluorescent staining intensity in transformed HaNIH as compared with NIH/3T3 cells. The marked similarities between the LAMP proteins raise the consideration of common functions, possibly associated with the high oligosaccharide content of the molecules.

HIV-mediated defects in immune regulation.

The Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases (NIAID), sponsored a Workshop on HIV-Mediated Defects in Immune Regulation on September 29-30, 1993. Workshop participants included investigators in basic research of immune regulation, animal models of HIV disease, HIV epidemiology, and HIV clinical research and treatment. The purpose of the workshop was to describe and evaluate biological mechanisms of HIV-mediated immune deficiency other than direct killing of infected CD4+ cells. The workshop focused on HIV-mediated dysfunction in signal transduction and in T cell development and maturation. Mechanisms by which HIV has been proposed to influence signal transduction include gp120 ligation to CD4, HIV superantigen(s), and HIV-mediated perturbations in signal pathway components (e.g., receptors, kinases, phosphatases, cytokines, and cyclins). As a result of signal dysfunction, cells may fail to respond to foreign antigens (anergy) or become predisposed to enter suicide pathways, otherwise known as programmed cell death or apoptosis. Programmed cell death is a normal immune regulatory mechanism that is activated to prevent anti-self responses and also to delete expanded but no longer needed cell populations. In the immune system, new cells are constantly produced from stem cells to replace those that die from age, pathological response, or programmed cell death. Dysfunction in these new cells may occur if HIV causes changes in the structural environment of the thymus and lymph nodes, or in cytokine signals.

Early phases of HIV type 1 infection.

A workshop entitled "Early Phases of HIV-1 Infection" was held to review current research on the immunological and virological aspects of early phases of HIV infection in humans and in animal models, to identify studies for future research, and to foster collaborations among investigators in the biomedical community. In infections of adults, the appearance of cytotoxic T lymphocyte activity, when present, coincides with a decrease in viral load as measured by plasma viremia. In neonatal infections, however, an initial decrease in viral load has been observed months before cytotoxic T lymphocytes are detected. Immunological data, from a limited number of patients, indicated that CD8+ cytotoxic T lymphocytes detected early after HIV-1 infection may recognize epitopes in any of several HIV-1 proteins: Env, Gag, Pol, Tat, and Nef. With regard to the humoral antibody response, anti-Env binding antibodies appear before neutralizing antibodies and do not predict the appearance of neutralizing activity. The time at which neutralizing antibody appears is variable and unpredictable. Preliminary data indicate that early viral peak load does not predict disease progression in many cases, and the phenotype or virulence of the virus appears to be a critical variable. However, the quantity of HIV-1 RNA in plasma is a strong CD4+ T cell-independent predictor of outcome following HIV-1 seroconversion in homosexual men. Early, high virus load with sustained viremia is often accompanied, in both adults and infants, by the inability to mount an effective immune response, resulting in rapid disease progression.

Neutralization of HIV-1.

A Workshop on Neutralization of HIV-1: Technology and reagents for analysis of prophylactic vaccines clinical trials, sponsored by the Food and Drug Administration (FDA) and the Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), was held on April 19-20, 1993, in Bethesda, Maryland. This workshop brought together researchers who are involved in the development, testing, and evaluation of HIV-1 prophylactic vaccines. The major objectives were (1) to discuss critically the different neutralization and binding assays that are currently used in the evaluation of immune sera; (2) to identify assays that will measure the "most relevant" antibodies, which are likely to predict neutralization of primary isolates; and (3) to identify well-characterized reference reagents, which could be used to standardize neutralization assays used in laboratories around the world.

International collaboration comparing neutralization and binding assays for monoclonal antibodies to simian immunodeficiency virus.

Thirteen laboratories characterized a coded panel of 10 MAbs to SIVmac251 envelope protein in a collaboration organized by the National Institute of Allergy and Infectious Diseases (NIAID). The MAbs were examined against SIV isolates in neutralization and radioimmune precipitation, immunoblot, enzyme-linked immunosorbent, and radioimmune assays. Although laboratories employed diverse neutralization assays that varied in sensitivity there was agreement on the relative ability of the MAbs to neutralize SIVmac251. Additionally, even though the quantity of any single MAb required to neutralize SIVmac251 varied between laboratories, there was agreement on the rank-order strength fo the five neutralizing MAbs. Based on the data from this study, the MAbs were classified according to their neutralization potential as high efficiency (MAb concentration, < 5 micrograms/ml), low efficiency (MAb concentration, 5-100 micrograms/ml), or nonneutralizing (MAb concentration, > 100 micrograms/ml). The MAbs could be assigned to four serological groups based on ability to cross-neutralize and bind different SIV isolates. The distinction between groups I, II, and III were based on the limited neutralization data obtained with the sooty mangabey isolate.