Colocalization of heparin and receptor binding sites on keratinocyte growth factor.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. FGFs are also known as heparin-binding growth factors because they bind to heparin and their physical and biological properties are modulated by heparin. Consistent with a role as a paracrine effector, KGF is produced by cells of mesenchymal origin but is active primarily, if not exclusively, on epithelial cells. KGF is involved in a variety of physiological processes, including proliferation, differentiation, wound healing, and cytoprotection. To identify regions in KGF that contribute to heparin and tyrosine kinase receptor interactions, nine peptides spanning defined motifs in the predicted structure of KGF were synthesized, and their heparin and receptor binding properties were analyzed. Peptides at the amino and carboxyl termini bound heparin, and one peptide showed relative binding comparable to that of KGF. Competitive binding studies showed that this peptide along with two other overlapping peptides specifically displaced KGF bound to the KGF receptor. These three peptides were also selectively recognized by a neutralizing monoclonal antibody against KGF, though only in the presence of heparin. Together, these data suggest that the sites for heparin and receptor binding both reside in the amino and carboxyl termini of KGF, which are spatially juxtaposed in the predicted three-dimensional structure of this molecule.

Mixed-backbone oligonucleotides as second generation antisense oligonucleotides: in vitro and in vivo studies.

Antisense oligonucleotides are being evaluated in clinical trials as novel therapeutic agents. To further improve the properties of antisense oligonucleotides, we have designed mixed-backbone oligonucleotides (MBOs) that contain phosphorothioate segments at the 3' and 5' ends and have a modified oligodeoxynucleotide or oligoribonucleotide segment located in the central portion of the oligonucleotide. Some of these MBOs indicate improved properties compared with phosphorothioate oligodeoxynucleotides with respect to affinity to RNA, RNase H activation, and anti-HIV activity. In addition, more acceptable pharmacological, in vivo degradation and pharmacokinetic profiles were obtained with these MBOs.

Nef protein of HIV-1 has B-cell stimulatory activity.

OBJECTIVE: To examine the B-cell stimulatory properties of the regulatory Nef protein of HIV-1. METHODS: The effect of the HIV-1 regulatory proteins Nef, Tat and Vif, were analyzed for their ability to induce differentiation of normal B lymphocytes into immunoglobulin secreting cells (ISC). RESULTS: A recombinant Nef protein, but neither Tat or Vif, was able to induce ISC in peripheral blood lymphocyte (PBL) cultures of HIV-1-seronegative donors. Another recombinant Nef protein, d-Nef, with a truncated amino terminal (deletion of 34 amino acids) failed to induce B-cell differentiation. Pretreatment of the Nef protein with a polyclonal anti-Nef-antibody abrogated its B-cell stimulatory activity. The Nef-induced B-cell differentiation was dependent on cell-to-cell contact. Cell surface molecules leukocyte function-associated molecule (LFA)-1, intracellular adhesion molecule (ICAM)-1, human lymphocyte antigen-DR and B7 were involved in the T-B-cell interaction because monoclonal antibodies to these molecules abrogated the Nef-induced B-cell differentiation response. The Nef protein was able to induce interleukin (IL)-6 messenger (m)RNA and IL-6 protein secretion in PBL, with monocytes as the primary source. CONCLUSIONS: These findings indicate that regulatory (Nef) proteins of HIV-1 contribute to the intense B-cell activation that occurs in association with HIV-1 infection. T-B-cell contact-dependent interaction and induction of IL-6 by these proteins appear to play major roles in this process.

Subunit interaction in B19 parvovirus empty capsids.

B19 parvovirus is a small single-stranded DNA virus with a genome that encodes only two structural proteins, designated VP1 and VP2. 60 copies of the structural proteins assemble into the viral capsid, with approximately 95% VP2 and 5% VP1. Recombinant empty capsids composed of VP2 alone or of VP2 and VP1 self-assemble into particles that are morphologically indistinguishable from full virions. Empty capsids containing both VP2 and VP1 elicit a strong neutralizing antibody response when used to immunize rabbits. Capsids containing only VP2 are similarly antigenic but elicit only weak neutralizing activity. We performed fine structure epitope mapping by measuring the reactivity of antisera raised against capsids composed of VP2 and VP1 or VP2 alone against 85 overlapping peptides spanning the sequence of the two structural proteins. A profile of the antigenic difference between empty capsids with and without VP1 was produced from the resulting data. This profile divided the sequence of the structural proteins into four regions that correlated well with expected viral structures. Thus, the addition of a small number of VP1 residues altered the antigenicity of the entire capsid. The major area of enhanced antigenicity is homologous to the spike of canine parvovirus, an area known to contain both neutralizing and host-range determinants. Our data are consistent with a model in which the unique region of VP1 is necessary for the virus to assume its mature capsid conformation.

Frequency of adult T-cell leukaemia/lymphoma and HTLV-I in Ibadan, Nigeria.

Sera from a small sample of adult blood donors, healthy school children and patients with lymphoma, leukaemia, non-haematologic cancer, congenital and inflammatory disorders from Ibadan, Nigeria were screened for HTLV-I antibody by an enzyme-linked immunoabsorbent assay and confirmed by investigational Western blot. Seventy-nine of 236 positively screened samples could not be tested for confirmation. Seropositive reactivity was observed in nine of 123 blood donors, and 3 of 46 healthy school children but banding patterns on Western blot were often sparse. Among non-Burkitt's non Hodgkin's lymphoma patients six of 30 were HTLV-I positive including four of four with clinical features of adult T-cell leukaemia (ATL). Other clinical conditions had a frequency of positivity indistinguishable from healthy donors. Western blot patterns ranged from strong with multiple bands, which were uncommon, to those with only p24 and p21 envelope positive which were frequent. Given the relative paucity of clinical ATL and the unusual Western blot patterns the true rate of HTLV-I infection may be lower than estimated. It is possible that a cross-reactive HTLV-I-like virus accounts for this pattern.

Autopsy findings in HIV-infected inner-city patients.

To assess the importance of the autopsy in HIV-1 infection, we retrospectively reviewed the autopsy reports of 70 HIV-1-seropositive patients at Howard University Hospital. Of the 58 patients with AIDS, the diagnosis of AIDS was made after autopsy in 24 (41%) cases. The lung was the most common site of AIDS-diagnostic diseases, and was affected in 90% of patients. Pneumocystis carinii infection was the most common AIDS-diagnostic disease, and was present in 50% of the AIDS patients. Thirty-eight percent of AIDS diagnostic diseases were diagnosed antemortem, including 15 of the 29 Pneumocystis carinii infections. Most of the AIDS-diagnostic diseases were disseminated at autopsy and two or more diseases were found in some organs. Overall, Pneumocystis carinii pneumonia was the most common cause of death, accounting for a mortality of 43% among AIDS patients. Bacterial infections were common and contributed to the mortality and morbidity of both AIDS and non-AIDS patients. Bacterial infection was the cause of death in 15 AIDS and 9 non-AIDS patients. The clinical cause of death concurred with the pathological cause in 53% of our cases.

Sequential measurement of beta 2-microglobulin levels, p24 antigen levels, and antibody titers following transplantation of a human immunodeficiency virus-infected kidney allograft.

A renal allograft recipient developed symptoms suggestive of AIDS. Serological studies revealed that the donor was positive for human immunodeficiency virus (HIV). Retrospective testing of stored sequential serum samples showed that the recipient was negative for HIV pretransplant; anti-p24 and anti-p41 antibodies appeared 10 and 49 days posttransplant, respectively. The recipient's serum beta 2-microglobulin levels were elevated 14 days posttransplant, with normal renal function, 35 days before the detection of anti-p24 antibody. p24 Antigen was detected for the first time 21 days posttransplant. In addition to p24 antigen, elevated serum beta 2-microglobulins may be a useful marker for HIV infection prior to seroconversion.

A chronic "postinfectious" fatigue syndrome associated with benign lymphoproliferation, B-cell proliferation, and active replication of human herpesvirus-6.

A 17-year-old, previously healthy woman developed an acute "mononucleosis-like" illness with an associated "atypical" pneumonitis, followed by years of debilitating chronic fatigue, fevers, a 10-kg weight loss, night sweats, and neurocognitive symptoms. Thereafter, her sister developed a similar but less severe illness. The patient developed marked, chronic lymphadenopathy and splenomegaly, with associated persistent relative lymphocytosis and atypical lymphocytosis and with thrombocytopenia. After 3 years of illness, a splenectomy was performed, which resulted in some symptomatic improvement, prompt weight gain, and resolution of all hematologic abnormalities. Serial immunologic studies revealed a strikingly elevated number of activated B lymphocytes and a T lymphopenia, which improved but did not return to normal postsplenectomy. No causal association was found with any of several infectious agents that could produce such a lymphoproliferative illness. However, both the patient and her sister had evidence of active infection with the recently discovered human herpesvirus-6. Seven years after the onset of the illness, the patient and her sister remain chronically ill.

Prevalence of antibodies to human immunodeficiency virus and to human T cell leukemia virus type I in transfused sickle cell disease patients.

The prevalence of the human immunodeficiency virus (HIV) antibody and the human T cell leukemia virus type I (HTLV-I) antibody was examined in 116 adults with sickle cell disease. Eighty-eight of them had received a mean of 18.6 transfusions of red blood cells between 1978 and 1985, and none was positive for the HIV antibody. Of 116 patients, 9 (7.8%) tested positive for HTLV-I antibodies. HTLV-I-positive patients were similar to those without HTLV-I antibody with respect to age, number of transfusions, and proportion of patients with greater than 40 transfusions. However, 3 of the 9 HTLV-I-positive patients came from West Africa or from the Caribbean, whereas this proportion was much lower (7/107) in the HTLV-I-negative group (x2, 7.564; P less than .01). Our analysis suggests that the risk of HIV infection in transfused sickle cell disease patients is low. Although HTLV-I antibodies in these patients may not be related to blood transfusions, it seems prudent to screen blood donors for HTLV-I infection.

The spectrum of clinical and laboratory findings resulting from human herpesvirus-6 (HHV-6) in patients with mononucleosis-like illnesses not resulting from Epstein-Barr virus or cytomegalovirus.

Recently, the morphologic, immunologic, and molecular makeup of a new virus designated human herpesvirus-6 (HHV-6) has been described. Because cell cultures of HHV-6-infected mononuclear cells showed prominent lymphocytic changes, it could be anticipated that mononucleosis-like illnesses or lymphoproliferative disorders would turn out to be manifestations of active HHV-6 infection. In the present study, blood samples from 27 patients previously categorized as having non-Epstein-Barr virus (non-EBV)/noncytomegalovirus (non-CMV) heterophil-negative mononucleosis-like illnesses were tested for IgM and IgG antibodies to HHV-6. Eight of these patients (30%) had serologic evidence of active HHV-6 infection. The clinical spectrum includes a short-lived febrile illness, mild cervical lymphadenopathy, laboratory data suggestive of active viral hepatitis in two patients, and a prolonged febrile illness in a single patient with previously documented positive anti-HIV serology. The viral studies revealed the presence of fourfold HHV-6-specific IgG titer increases by immunofluorescent assay (IFA) in seven serially studied cases and positive IgM serology on one or more samples tested by IFA or enzyme-linked immunosorbent assay (ELISA) in all eight cases. The authors could not determine whether the illnesses represented primary HHV-6 infections in susceptible individuals or reactivation of latent virus. HHV-6 serologic studies may be indicated in patients with mononucleosis-like illnesses with atypical lymphocytosis when EBV and CMV test results are nondiagnostic.

Examination of HTLV-I ELISA-positive leukemia/lymphoma patients by western blotting gave mostly negative or indeterminate reaction.

We used enzyme-linked immunosorbant assay (ELISA) and Western blotting, with "purified" human T-cell leukemia virus I (HTLV-I), to test for HTLV-I antibodies in 2583 plasma samples from 1053 leukemia/lymphoma patients treated at Roswell Park Memorial Institute, mostly between 1972 and 1984, and in 110 sera samples from normal healthy persons. The results demonstrate that ELISA and Western blot assay have limitations for HTLV-I antibody detection in an adult T-cell leukemia/lymphoma (ATL) nonendemic population. This conclusion is based on the many false reactives obtained by ELISA, and weak and indeterminate reaction (mostly p19 band) on Western blotting. All moderate to strongly HTLV-I ELISA-positive samples tested were negative for human immunodeficiency virus (HIV) antibodies. Although 6/27 mycosis fungoides (MF) patients tested gave mostly a weak reaction on HTLV-I ELISA, 3/6 MF patients gave multiple bands (p19, p31, p36, gp46) on Western blotting and three samples from one patient gave the same p31, p36, and gp46 bands. This may suggest involvement of some HTLV-I-related virus in MF. These results also indicate that prevalence of HTLV-I infection in leukemia/lymphoma patients was rare, if it exists at all, since, despite the reactivity of some sera with HTLV-I-suspected antigens, none of the samples satisfy the USPHS criteria for positivity which is based on the detection of antibodies to gag protein p24 and to an env gene product gp46 or gp61/68.

Localization of B-cell stimulatory activity of HIV-1 to the carboxyl terminus of gp41.

Patients with AIDS are known to have B-cell hyperactivity. We have previously demonstrated that an extract of HIV-1 could induce differentiation of peripheral blood B lymphocytes of healthy volunteers into immunoglobulin-secreting cells. In an attempt to delineate the B-cell stimulatory subregion in HIV-1, we have investigated the influences of native glycoproteins and recombinant proteins of the envelope. The complete envelope glycoprotein, gp160 and a recombinant protein in the carboxyl terminal region of gp41 termed PE-8 were effective in inducing terminal differentiation of normal peripheral blood B lymphocytes and did so in a T-lymphocyte-dependent manner. The activity was not present in the native exterior envelope glycoprotein, gp120 and several other recombinant proteins, viz PE-2 an PE-3, which are in the amino terminal region of gp120 or in env-9, a protein in the junctional region of gp120 and gp41. Polyclonal and monoclonal antibodies directed to diverse regions of the envelope abrogated the influence of gp160. The PE-8-induced B-cell differentiation was abrogated by goat anti-gp160 antibody but not by goat anti-gp120 antibody or monoclonal antibody to the amino terminal of gp41. These studies suggest that a putative polyclonal B-cell stimulatory epitope of HIV-1 is located in the carboxyl end of the envelope glycoprotein.

A study of HTLV-I and its associated risk factors in Trinidad and Tobago.

Seroprevalence of human T-lymphotropic virus type 1 (HTLV-I) among a sample of persons selected from a government register of businesses in Trinidad was 3.2% in 1,025 persons of African descent compared to 0.2% among 487 persons of Asian descent and 0% among 46 persons of European-descent. In Tobago, from a coastal village, among persons of African ancestry ascertained as part of a cardiovascular survey, the rate was 11.4%, which was significantly higher when corrected for age and race than the rate in Trinidad. The seroprevalence rate of antibodies to hepatitis A and B was also significantly elevated in Tobago compared to Trinidad. HTLV-I seroprevalence rates were higher in females than males while hepatitis A and B rates were not significantly different in the two sexes. For males, age was a significant determinant of HTLV-I seropositivity, while for females, age, markers of poor sanitation, and hepatitis B were each independently linked to HTLV-I seropositivity. The frequent occurrence of multiple infectious exposures in persons of lower socioeconomic circumstances in this tropical environment may result in immune activation that heightens susceptibility to HTLV-I infection.

Serological confirmation of human T-lymphotropic virus type I infection in healthy blood and plasma donors.

We wished to develop criteria for serological confirmation of human T-lymphotropic virus type I (HTLV-I) infection in healthy donors. Selected serum or plasma samples reactive by HTLV-I enzyme immunosorbent assay or gel-agglutination assays with at least one viral-specific band on Western immunoblot (WIB) were tested in six laboratories by four WIBs and four radioimmunoprecipitation assays (RIPAs) for antibodies to HTLV-I proteins encoded by gag (p19 and p24), env (gp46 and/or gp61), and tax (p40x) genes. One hundred forty-two donor sera were obtained from 38 Japanese, 69 American, and 35 Caribbean blood or plasma donors. Among these samples, WIB assays appeared more sensitive to p24 antibodies, whereas RIPAs were significantly more sensitive to gp61 antibodies. All sera (137) with gp61 antibodies had p24 antibodies. Of the 137 sera positive for p24 and gp61 antibodies, p19 antibodies were detected in 129 sera, and p40x antibodies were detected in 108. In sera with p19 antibodies and antibodies to env- or tax-encoded proteins, p24 antibodies were always present. Antibodies to p40x were not found in the absence of gp61 antibodies. Virological evidence of infection was found in seven American donors by lymphocyte coculture (one HTLV-I, one HTLV-II) or by polymerase chain reaction (three HTLV-I, two HTLV-II). Sera from all seven donors showed p24 and gp46 and/or gp61 antibodies. We suggest that seroreactivity to both p24 and gp46 and/or gp61 by WIB or RIPA or both are suitable criteria to confirm but not to distinguish HTLV-I and HTLV-II infections.

No evidence for true HTLV-I or HIV-1 antibodies in Finnish Lapps.

Previous seroepidemiologic studies have suggested that in addition to certain subtropical and tropical parts of the world, human T cell leukemia virus type I (HTLV-I) may be endemic in the arctic regions, too. We studied 111 sera collected from original inhabitants of Finnish Lapland with ELISA and Western blot analysis for antibodies to HTLV-I and human immunodeficiency virus type 1 (HIV-1). No true positive sera for either virus were found in the confirmatory Western blot assays, albeit 6 and 2%, respectively, were positive in the screening ELISA assays. Despite the small sample size this survey does not support the hypothesis that HTLV-I would be endemic in the Arctic.

Influences of related retroviruses on lymphocyte functions.

The human immunodeficiency virus (HIV-1) is known to be profoundly immunosuppressive [Spickett and Dalgleish (1988) Clin. Exp. Immunol. 71, 1]. In this communication, we have studied the influences of HIV-1 (BH10), HIV-2 (LAV-2) and STLV-3 on B and T cells from healthy volunteers. B lymphocytes were found to differentiate into immunoglobulin secreting cells in response to stimulation by proteins of HIV-1 and LAV-2, but not by STLV-3. This response was obtained at protein concentrations of 0.05-0.005 micrograms/ml and was T cell dependent. IgM secretion was induced only by HIV-1 in the EBV-transformed B cell line SKW 6.4. At higher concentrations all three retroviral preparations had inhibitory influences on functions of B as well as T lymphocytes. B cell differentiation was maximally inhibited by HIV-1 and LAV-2 when these proteins were added concurrently to cultures with the polyclonal B cell activators pokeweed mitogen or Epstein-Barr virus. Tetanus antigen-specific T cell lymphoproliferation was inhibited by all retroviral proteins. These findings suggest that related retroviruses differ in their capacity to influence normal immune responses.

A historical study of human T lymphotropic virus type I transmission in Barbados.

A 1972 historic sera collection from two health districts in Barbados, British West Indies, was evaluated for risk factors and epidemiologic patterns of HTLV-I (human T cell leukemia virus type I) during a time prior to the first report of its discovery in 1980. HTLV-I seroprevalence is 4.2% (43 of 1,012) and is consistent with current estimates in endemic areas in the Caribbean. Age-dependent rise (P less than .01) and higher seroprevalence rates for females (P less than .01) are indistinguishable from the pattern in contemporary Caribbean and Japanese populations. HTLV-I seropositivity was 4 times higher in women (P less than .003) and 2.6 times higher in men (P = .32) with treponemal antibodies, supporting a role for sexual transmission. Children who were positive in a household were more likely to have a seropositive mother than a seropositive father. This pattern is consistent with transmission of the virus from mother to child. Our results document that rates of infection and modes of transmission of HTLV-I are persistent.

In vitro inhibition of human herpesvirus-6 by phosphonoformate.

HSB-2 cell cultures productively infected with human herpesvirus-6 were treated with the antiviral drugs phosphonoformic acid (PFA), acyclovir (ACV), and gancyclovir (DHPG). ACV and DHPG showed significant toxic effects on uninfected HSB-2 cells, yet only incompletely inhibited viral expression upon infection of the cells. PFA, however, showed little direct toxicity on HSB-2 cells while viral replication was inhibited significantly.