Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia.

Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25-FoxP3-LAP+ T cells (but not of CD4+CD25+FoxP3+ T cells) in the lamina propria of the small but not large intestine. The adoptive transfer of very small numbers of CD4+CD25-LAP+ Treg isolated from the spleen of tolerized mice was superior in suppression of antibodies directed against FIX when compared to CD4+CD25+ T cells. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine, and suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg that likely migrate to the spleen. Tolerogenic antigen presentation in the small intestine requires partial enzymatic degradation of plant cell wall by commensal bacteria in order to release the antigen. Microbiome analysis of hemophilia B mice showed marked differences between small and large intestine. Remarkably, bacterial species known to produce a broad spectrum of enzymes involved in degradation of plant cell wall components were found in the small intestine, in particular in the duodenum. These were highly distinct from populations of cell wall degrading bacteria found in the large intestine. Therefore, FIX antigen presentation and Treg induction by the immune system of the small intestine relies on activity of a distinct microbiome that can potentially be augmented to further enhance this approach.

Bacterial endocarditis in a child with haemophilia B: risks of central venous catheters.

The use of central venous catheters may be complicated by thrombosis and infection. We report a case of a needle-phobic 5-year-old boy with factor IX deficiency, in whom a portacath was inserted owing to poor compliance with prophylactic treatment. Within a week, he developed a Staphylococcus aureus line infection that was treated with a 2-week course of intravenous antibiotics. One month later he presented with nonspecific symptoms and blood cultures again grew S. aureus. An echocardiogram revealed a large vegetation adherent to the tricuspid valve, confirming the diagnosis of bacterial endocarditis. His clinical course was further complicated by the development of pulmonary emboli. Medical treatment with intravenous antibiotics led to a successful resolution of the endocarditis and pulmonary emboli with a favourable long-term outcome.

Hepatitis C virus-RNAs in plasma and in peripheral blood mononuclear cells of hemophiliacs with chronic hepatitis C: evidence for viral replication in peripheral blood mononuclear cells.

Hemophiliacs who have been exposed to unheated and/or dry heated pooled clotting factor concentrates are at high risk of chronic hepatitis C. Although the mechanism and site of hepatitis C virus (HCV) replication are not yet known, HCV is thought to replicate through a complementary negative RNA strand, as has been shown for flaviviruses. The detection of negative RNA strands has therefore been regarded as a marker of replication. We investigated the prevalence of HCV-RNA and of negative HCV-RNA strands in peripheral blood mononuclear cells (PBMC) and plasma of hemophiliacs. Forty-three of 47 patients studied (91%) had anti-HCV antibodies and in 36 patients HCV-RNA was detectable in PBMC. In one group of 20 patients negative HCV-RNA strands were present in PBMC and 10 of these patients also had negative HCV-RNA strands in plasma. In another group of nine patients HCV-RNA was detected in PBMC, although cDNA synthesis was carried out in the absence of primers. Only in two of these nine patients negative and positive HCV-RNA strands were demonstrated specifically in PBMC using a modified reverse transcription step. If the presence of negative HCV-RNA strands can be considered as marker of viral replication, the findings indicate that HCV can replicate in PBMC. Furthermore, in certain patients it is impossible to use the currently available technique to detect selectively positive or negative HCV-RNA strands.

Six strains of human immunodeficiency virus type 1 isolated in Japan and their molecular phylogeny.

Five strains of human immunodeficiency virus type 1 (HIV-1) were isolated from five Japanese hemophilia patients. Two isolates, HIV-1[GUN-1] and HIV-1[GUN-2], were from brother patients with hemophilia B and the other three isolates, HIV-1[GUN-3], HIV-1[GUN-4], and HIV-1[GUN-5], were from hemophilia A patients. Another HIV-1 strain, HIV-1[GUN-6], was isolated from a Canadian male homosexual with AIDS. The restriction endonuclease cleavage maps of the proviral genomes of these six HIV-1 strains revealed that they were apparently different from each other. The phylogenetic trees constructed using restriction maps and nucleotide sequences were quite similar, indicating that phylogenetic analyses of Japanese HIV-1 isolates can be done using restriction maps of the proviruses. Phylogenetic analyses showed that they were more closely related to HIV-1s which had been reported to be isolated from homosexual patients in the United States than those isolated from African patients. In particular, GUN-1 and GUN-2 isolates were on the branch of a San Francisco isolate, ARV2, while GUN-5 and GUN-6 isolates were on the branch of HTLV-IIIB-related isolates.

Epidemiologically closely related viruses from hemophilia B patients display high homology in two hypervariable regions of the HIV-1 env gene.

The diversity of human immunodeficiency virus type 1 (HIV-1) is mainly caused by mutations that affect the gene encoding the gp120 envelope protein. Isolates differ to a large extent in the hypervariable regions of gp120. This study was undertaken to determine the degree of variation of HIV-1 env genes isolated from seven individuals with hemophilia B who became infected in association with administration of a suspected clotting factor lot. Two hypervariable regions and part of a constant region from proviral DNA of the peripheral blood leukocytes of these patients were amplified and the products of the polymerase chain reactions were sequenced. The sequences derived from five of the individuals displayed 100% sequence homology, 1 had two and 1 had six deviations from the consensus sequence. The alignment of the amino acid sequence so deduced revealed no comparable homology to any of these two hypervariable regions from a number of published isolates. The genetic variability of HIV-1 seems to be limited, at least in the early phase of infection, allowing the determination of close relationships between epidemiologically related strains.

Immunological and virological status of a hemophiliac infected with human T cell lymphotropic virus type 1 and human immunodeficiency virus type 1, and results of therapy.

The immunological and virological status of three hemophiliacs infected with human immunodeficiency virus type 1 (HIV-1) was monitored for 11 months. One of these patients was also infected with human T cell lymphotropic virus type 1 (HTLV-1), and HIV-1 could be isolated only from this patient among the three subjects. The doubly infected subject had the fewest T4 (helper) lymphocytes and the highest proportion of T8 lymphocytes with DR human leukocyte antigens (DR-Ag). The serum level of HIV-1 antigen increased in this patient during the observation period, and this increase was accompanied by a decrease in the proportion of DR-Ag-positive cells among the T8 lymphocytes. This patient was treated with 800 mg of zidovudine daily for 50 days. With treatment, the nonspecific clinical symptoms improved and the proportions of DR-Ag positive cells among the T8 lymphocytes decreased. Serum levels of HIV-1 antigen decreased immediately when therapy was started but later increased during therapy. In persons infected with both HTLV-1 and HIV-1, HIV-1 seems to proliferate readily.

Isolation of human immunodeficiency virus from hemophiliacs: correlation with clinical symptoms and immunologic abnormalities.

As part of a prospective study of human immunodeficiency virus (HIV) infection in hemophilia, peripheral blood mononuclear cells (PBMs) from 72 individuals without acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) were cultured for virus. HIV was isolated from PBMs from 16 (24%) of 66 patients with hemophilia who were seropositive for HIV and from none of six seronegative patients. Cells from five of six patients from which HIV was isolated were again successfully cultured for virus 3 to 12 months later. HIV core P24 antigen was detected in serum from seven of 15 patients with HIV-positive cells and from eight of 50 with HIV-negative cells. Patients with hemophilia with isolation-positive cells had significantly fewer T helper cells and significantly lower T helper/T suppressor ratios, pokeweed mitogen responsiveness, and total platelet counts than did those whose cells did not yield HIV on cultivation. HIV neutralizing antibody titers did not differ between hemophiliacs with or without HIV-positive PBMs. Three of the 16 patients with virus-positive cells developed AIDS, and two ARC, within 18 months of the study, compared with three of 50 seropositive hemophiliacs whose cells did not yield virus, who developed ARC during the same period. The significant decrease in the number of T helper cells, decreased platelet counts, and higher rate of progression to AIDS in the group with HIV isolation may reflect a heavier virus load, indicating that the ability to culture HIV may be an early marker of more significant disease.

Antibody to human immunodeficiency virus in factor-deficient plasma. Effect of heat treatment on lyophilized plasma.

The prevalence of antibody to human immunodeficiency virus (HIV) was determined in various commercial substrate plasmas used in clotting factor assays, and viral isolation was attempted from both seropositive and seronegative samples. Antibody to HIV was detected in 13 of 13 plasma substrates used for Factor VIII assays and in 2 of 3 plasma substrates used for Factor IX assays. Antibodies were not detected in any of the other factor-specific substrates. Virus could not be isolated from any of the seropositive samples after 28 days in culture. Heat treatment of the samples under conditions known to inactivate HIV in plasma products indicated that heating the lyophilized substrate plasmas at 60 degrees C for up to eight hours had little effect on factor substrates and factor assays. Progressive loss of Factor V in the deficient plasmas was the most serious effect produced by heat treatment.

Isolation of human immunodeficiency virus from a Japanese hemophilia B patient with AIDS.

Human immunodeficiency virus (HIV) was isolated from a Japanese hemophilia B patient with AIDS. This isolate, HIV[GUN-1], was infectious to several mature T-cell lines. Proteins with apparent molecular weights of 160, 55 and 25 kilodaltons were detected. Restriction enzyme cleavage patterns of the proviral genome indicated that HIV[GUN-1] is related to but clearly different from HTLV-III or ARV-2.

Prevalence of HTLV-III/LAV antibodies in Italian asymptomatic hemophiliacs given commercial concentrates of factors VIII and IX.

The prevalence of HTLV-III/LAV antibodies was evaluated in 222 Italian asymptomatic hemophiliacs treated exclusively with commercial factor concentrates made with American plasma. An increase of HTLV-III/LAV seropositivity from 1983 to 1985 was evident. This was independent of the type of hemophilia (A or B) but directly correlated to the amount of concentrate administered in the previous year. Immunological data (T4 and T8 lymphocyte subsets) were evaluated in relation to seropositivity to HTLV-III/LAV. The presence of antibodies was found to be significantly associated with a low T4/T8 ratio and to a reduced T4 subpopulation, whereas increased levels of T8 lymphocytes correlated weakly with seropositivity. Data from 47 hemophiliacs tested both in 1984 and 1985 showed that 100% of those negative in 1984 and highly transfused in the previous year seroconverted up to 1985, whereas 33% of those not highly transfused did so.

Prevalence of antibodies to lymphadenopathy-AIDS virus in French haemophiliacs.

49 French haemophiliacs (haemophilia A: 41 patients; haemophilia B: 8 patients) were serologically testes for lymphadenopathy-AIDS virus antibodies: 10 patients (20.4%) were seropositive including 9 (21.9%) with haemophilia A and 1 (12.5%) with haemophilia B. Among haemophiliacs A, seropositive patients received significantly larger amounts of factor VIII concentrate during the 2 years preceding the study.

Isolation of new lymphotropic retrovirus from two siblings with haemophilia B, one with AIDS.

A human T-lymphotropic retrovirus was isolated from cultured T lymphocytes from two siblings with haemophilia B. Patient 2 was healthy, but patient 1 had acquired immunodeficiency syndrome. The retrovirus differed from human T-cell leukaemia virus (HTLV) but it was similar to the lymphadenopathy-associated retrovirus (LAV) in its morphology and its major core protein (P25). Both patients had antibodies against LAV and patient 1's retrovirus, detected by an enzyme-linked immunosorbent assay or a radioimmunoprecipitation assay. Seroepidemiological data indicated the transmission of this retrovirus by plasma products.

Serologic evidence of hepatitis B virus infection in patients with hemophilia B. a multicenter study.

Among sera from 160 patients with hemophilia B from 9 centers in Europe and North and South America, 2.5% were positive for hepatitis B surface antigen (HBsAg), 60for antibody to HBsAg, and 31% for antibody to the hepatitis B core antigen. Evidence of exposure to the hepatitis B virus appeared to be related to severity of disease and age rather than the source and method of manufacturer of factor IX concentrate.