Human T-cell heterogeneity as delineated with a specific human thymus lymphocyte antiserum. In vitro effects on mitogen response mixed leukocyte culture, cell-mediated lymphocytotoxicity, and lymphokine production.

Human peripheral blood lymphocytes (PBL) were evaluated by their responses to phytohemmagglutinin (PHA-P), concanavallin A (con-A), and pokeweed mitogen (PWM), both before and after treatment with an antiserum against human thymic lymphocyte antigens (HTLA) that had been made T-cell-specific by multiple absorptions with immunoglobulin EAC-positive lymphoblast cell lines (B cells). Cells treated with HTLA were examined for their ability to react in a mixed lymphocyte culture (MLC) and to form killer cells in a cell-mediated lymphocytotoxicity (CML) system. Sensitized cells were also examined for their ability to respond to purified protein derivative (PPD) by blastogenesis, migration inhibitory factor release (MIP), and lymphotoxin (LT) production, both before and after treatment with HTLA and complement. The HTLA was in itself highly stimulatory to PBL. However, with the addition of complement and subsequent cell destruction, a marked decrease in its stimulatory response was noted. PBL treated with HTLA and complement exhibited marked inhibition of responsiveness to con-A with little decrease in PHA-P -OR PWM stimulation except at very high concentration of HTLA. MLC reaction was inhibited only when responder cells were treated with HTLA + C'. Treatment of stimulator cells with HTLA + C' did not significantly alter the MLC response. The HTLA + C'-treated cells failed to form killer cells in the CML reaction and inhibited PPD-induced blasto-genesis from PPD-sensitized individuals; however, treatment of sensitized cells with HTLA + C' had little effects on the release of MIF and LT. It is suggested that subpopulations of T-cells carry surface antigens that bind with this specific antisera, and that the con-A-responsive cells, the responder cells in the MLC, and killer T-cells comprise a separate subset from cells responding to PHA-P or PWM, OR THE MIF-and LT-producing cells.

Sensitive method for detecting viable cells seeded into bone marrow.

A method for marking viable cells which allows highly sensitive detection of the cells in mixed populations is described. Viable tumor cell lines or normal lymphocytes were stained with the supravital DNA stain Hoechst 33342 (H342) and seeded into human bone marrow. Examination of the marrow mixtures with an inverted fluorescence microscope allows detection of a single H342-stained cell in 1 million marrow cells. H342 staining does not cause significant changes in cell viability or in the sensitivity of the cells to complement mediated lysis. Counterstaining the mixture with trypan blue quenches H342 fluorescence in nonviable cells, limiting detection to only viable cells.

Formation of tubuloreticular inclusions in human lymphoma cells compared to the induction of 2'-5'-oligoadenylate synthetase by leucocyte interferon in dose-effect and kinetic studies.

Exposure of human lymphoblastoid cell cultures to leukocyte interferon initiated the formation of membranous tubuloreticular inclusions (TRI) within the endoplasmic reticulum or perinuclear envelope. In four different cell lines originating from patients with lymphocytic cancers (Daudi, Raji, H-SB2, and SB), this unique ultrastructural effect displayed a log-linear relationship to increasing doses of interferon-alpha and was dose and time dependent. TRI morphogenesis began within 12 hr in Daudi or Raji cells exposed continuously to 500 IU of leukocyte interferon/ml, but only at 20 hr after 2- to 4-hr pulse exposures to 50 to 100 IU/ml. The TRI accumulation, determined by thin-section counts, reached maximal levels of up to 16% of cell sections within 48 to 72 hr. Experiments with Raji cells indicated a decrease in TRI formation during successive cell divisions; detection of TRI after a pulse of interferon was enhanced when DNA replication was arrested. TRI morphogenesis appeared to be independent of several other known biological actions of interferons. It manifested later than the induction of 2'-5'-oligoadenylate synthetase activity which has been correlated with establishment of the antiviral state and occurred in cell lines, including DaudiKIFNR, which resist the growth-inhibitory effects of leukocyte interferon. Participation of new polypeptide synthesis was indicated by experiments with inhibitors of transcription (actinomycin D) or translation (cycloheximide): TRI morphogenesis was blocked when actinomycin D was added 4 hr after interferon and was reduced when cycloheximide was added for the interval of 13 to 23 hr after interferon.

Diagnostic usefulness of five screening assays for HIV in an east African city where prevalence of infection is low.

Five commercial screening assays for HIV-1, evaluated for their usefulness in detecting infection in high-risk groups in the East African country of Djibouti, produced varying degrees of performance when compared to Western blot and immunofluorescence confirmatory assays. In this population with a low prevalence of HIV infection (16/599), two enzyme-linked immunosorbent assays (ELISA; Abbott and Elavia) and two rapid assays (cambridge latex agglutination and Du Pont's HIV-CHEK) exhibited less than optimal sensitivities. However, with the exception of Elavia, these assays displayed excellent specificities. The fifth test (Serodia gelatin agglutination) produced the highest sensitivity (0.938) and negative predictive value but the lowest specificity and positive predictive value of all five tests. False positive reactions, which occurred only with the Elavia and Serodia tests, could not be explained on the basis of early infection in patients or cross-reactions with the related retroviruses HIV-2 and HTLV-I. We conclude that none of the five tests alone is sufficient in this testing situation, but that a combination of tests could satisfy most requirements for identifying HIV-1 reactive and non-reactive sera.

HIV-1 and hepatitis B transmission in Sudan.

A seroepidemiologic survey was conducted among 773 male soldiers living in five urban locations in Sudan to study the prevalence of and risk factors for HIV-1 and hepatitis B transmission. Twenty-eight per cent of the study population were born and raised in southern Sudan, an area bordering Kenya, Zaire and Uganda, whilst 72% of the study subjects were from northern Sudan. Seventy-eight per cent of the study population had serologic evidence of past hepatitis B infection, and 13 soldiers were confirmed positive for HIV-1 antibody. All 13 HIV-positive soldiers had recently been deployed in southern Sudan. Multivariate analysis indicated an association between living in southern Sudan and both hepatitis B (odds ratio 8.2) and HIV-1 infection (odds ratio 14). Additionally, sexual relations with prostitutes (odds ratio 1.5) and medical injections for schistosomiasis (odds ratio 2.72) were independent predictors of hepatitis B markers in this military population. The findings of this study suggest that sexual promiscuity is a risk factor for hepatitis B transmission in Sudan. They also indicate one possible route for the spread of HIV-1 from central to northern Africa.

Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2.

There is evidence that treatment with an antibody to tumor necrosis factor alpha (TNF alpha) improves an animal model of multiple sclerosis (MS) and is beneficial in two systemic inflammatory disease in humans, but there are no reports about anti-TNF treatment of MS. Therefore, we treated two rapidly progressive MS patients with intravenous infusions of a humanized mouse monoclonal anti-TNF antibody (cA2) in an open-label phase I safety trial and monitored their clinical status, gadolinium-enhanced brain magnetic resonance imaging (MRI), and peripheral blood and cerebrospinal fluid (CSF) immunologic status. We did not notice any clinically significant neurologic changes in either patient. The number of gadolinium-enhancing lesions increased transiently after each treatment in both patients. CSF leukocyte counts and IgG index increased after each treatment. The transient increase in the number of gadolinium-enhancing lesions that followed each infusion of cA2 together with the increase in cells and immunoglobulin in the CSF of each patient suggest that the treatment caused immune activation and an increase in disease activity. These results suggest that further use of cA2 in MS is not warranted and that studies of other agents that antagonize TNF alpha should be carried out with frequent monitoring of gadolinium-enhanced MRIs.

Characteristics of the 'active rosette test'. I. Technical considerations of the test and comments.

Using normal subjects, it was found that the 'Active Rosette Test' is both reproducible and consistent if performed exactly as described by Wybran et al. (1972). Variations in incubation time, centrifugal force while sedimenting rosettes, resuspension time after centrifugation, SRBC lymphocyte ratio, and increasing concentrations of fetal calf serum all produced marked deviations in test results. Rosette inhibition studies using a specific anti-T cell antisera showed inhibition of 'active' and total rosettes at the same level, suggesting that there are no marked variations in surface T antigens among respective T cells. Unless differences in functional activities can be demonstrated between the 'active' rosette-forming cells and the other T cells, the test will be of an empiric nature.

Harvest of human bone marrow directly from bone.

Harvest of human bone marrow directly from freshly resected bone provides purer preparations of marrow than can be obtained by the conventional technique of multiple aspirations from the iliac crests. In particular, directly harvested marrow is much less heavily contaminated with peripheral blood lymphocytes, a known source of mature T cells. Because of the possible relevance of these contaminating T cells for cadaveric bone marrow transplants, the best source of human marrow harvested directly from bone has been studied. Human bone marrow was harvested from 46 surgical specimens and 9 cadaveric tissue donors. Vertebral bodies provided the best source of bone marrow with average yields of 3.1 +/- 1.6 X 10(9) cells per vertebra. When entire ilia were removed and processed for marrow, an average of 1.6 +/- 1.0 X 10(9) cells was obtained. Surgically resected ribs yielded lower amounts of marrow with a mean cell number of 3.2 +/- 2.6 X 10(8) per rib. Isolation of bone marrow mononuclear cells from these preparations by density gradient centrifugation resulted in a loss of 45% of the starting cells. Human bone marrow was found to contain 5-6% T cells before gradient separation and these cells were immunologically competent as measured in vitro by responses to mitogens and alloantigens. This technique may be useful in obtaining human bone marrow for both immunologic studies and allogeneic transplantation.

Stability studies on horse antithymocyte globulin.

Two equine antithymocyte globulin preparations were studied with respect to their relative rosette inhibition activity after storage at 4 and 25 C. After storage at 4 C for 48 months no activity loss was observed; 25 C storage resulted in fragmentation and eventual loss of relative rosette inhibition activity. A third preparation was studied by monkey skin graft prolongation. During a 1-year period, there was no loss of immunosuppressive activity as measured by this method and the rosette inhibition assay.

Monoclonal and xenoantibodies specific for HLA-DR inhibit primary responses to HLA-D but fail to inhibit secondary proliferative (PLT) responses to allogeneic cells.

Monoclonal antibodies (DA-2 and CA-2) and xenoantisera (rabbit anti-human, p23,30) specific for HLA-DR framework determinants were added to primary and secondary mixed lymphocyte cultures. Although such antisera were shown to inhibit primary MLC, primed lymphocytes were much less sensitive to the blocking effects of these antibodies. In the studies shown here, the concentration of antibody required to inhibit primary MLC reactions was 0.1-1.5 micrograms/ml).

Antigen-specific human T-lymphocyte clones. Genetic restriction of influenza virus-specific responses to HLA-D region genes.

Human T lymphocytes, primed in vitro to influenza virus, were cloned by limiting dilution and expanded using medium containing interleukin 2 and feeder cells. A detailed analysis of the genetic requirements for induction of T-cell proliferation was conducted using a panel of cells from unrelated donors and two families who had previously been extensively phenotyped for HLA region antigens. Clones obtained from a Dw1, Dw3 individual required Dw1,DR1 histocompatibility for successful presentation of viral antigens by antigen-presenting cells. The antigen-presenting ability segregated with HLA-B,D,DR in an informative HLA-A/B recombinant individual. In contrast, some TLCs responded to antigen presented by cells that did not share known HLA antigens, and in one informative family, reactivity did not segregate with HLA. None of the T-cell clones reacted to allogeneic cells in the absence of antigen, suggesting that the TLCs do not bear receptors that recognize both influenza virus and alloantigen. In antibody-blocking studies, Dw1, DR1-restricted clones were blocked by all monoclonal anti-DR framework antibodies. The non-HLA-restricted TLCs were blocked by some, but not all, of the anti-DR framework monoclonal antibodies. These results confirm and extend the role of HLA-D region gene products in antigen presentation and also provide evidence that yet undefined cell interaction products, which may include hybrid structure, are able to participate in antigen-specific proliferative responses by human T cells.

Induction of antibody responses in vivo by antigen specific helper factor.

T helper cell factors (HF) have been preparated against protein and synthetic antigens by restimulating in vitro induced helper cells with small amounts of antigen. HF when tested in vitro are antigen specific and capable of replacing T cells, initiating a thymus dependent IgM response. In the in vivo adoptive transfer assay HF is capable of replacing T helper cells and is active at very high dilution, inducing both IgM and IgG responses. When tested on unirradiated DNP primed animals the HF was able to initiate a thymus dependent anti-hapten response, comparable to that seen with helper T cells, of both IgM and IgG class. It was also shown that HF acts on anti-Thy 1 treated spleen cells. The results indicate that antigen specific helper factors may be one of the physiologic mediators of T-B interactions in intact animals.

Comparison of antigen-specific I-region-associated cell interaction factors.

Two basic types of factors reacting with anti-I region (anti-Ia) antisera are compared, those derived from macrophage-like antigen presenting cells and others derived from T-lymphocytes, of either the suppressor or helper type. Despite the common property of reacting with anti-Ia antisera, the two sets of factors differ by many criteria. Macrophages, upon culture with antigen, release complexes of Ia antigen and a fragment of the original immunogen. This material is only produced by responder macrophages and thus appears to be a soluble Ir gene product. The genetic restriction of the T-macrophage interaction was investigated in chimeras, and it was found that the host environment as well as the donor genotype was of importance in determining restrictions, which were thus not really directed to "self." There was no evidence for intrinsic T-cell Ir genes, as nonresponder stem cells developed into responder T-cells in a (responder X nonresponder) F1 environment. However, these cells only responded in the presence of responder macrophages. Specific T-cell factors are different in nature. These all react with anti-Ia antisera, but the nature or function of the T-cell Ia is unknown. The basic structure involves a VARIAble region" responsible for antigen binding which, as it reacts with anti-idiotype antisera and anti-variable region framework antisera is an immunoglobulin variable region. There is also a "constant region," defined by its biological properties as well as by specific rabbit antisera. This two-region nature of specific factors is reminiscent of immunoglobulin structure and it is a reasonable hypothesis that the constant region is linked to the Ig cluster of genes.

T lymphocyte subset modulation of hepatic fibroblast function in murine schistosomiasis.

We have previously shown that co-culture of T cell enriched spleen lymphocytes can reduce collagen synthesis and stimulate proliferative activity by liver fibroblasts from S. mansoni infected mice. The present study examines which subset of T cells is responsible for this modulation. Co-culture of fibroblasts with the total T cell population lead to significant stimulation in fibroblast proliferative activity and a significant decrease in the incorporation of 14C-proline into collagenase-sensitive protein. There was virtually no effect on total incorporation of 14C-proline in non-collagen proteins. An additional significant increase in fibroblast proliferative activity and another significant decrease in collagen synthesis accompanied by a 2-fold increase in non-collagen protein production was noted when fibroblasts were co-cultured with Lyt-1+ cells. Co-culture of fibroblasts with Lyt-2+ cells did not differ significantly from co-culture with total T cells. Mitomycin treatment of the lymphocytes blunted their specific effects, suggesting that proliferation of T cells is required for maximal exertion of their regulatory activity. These results suggest that the T cells which mediate these effects belong to the Lyt-1+ helper class and are distinct from the Lyt-2+ suppressor cells.

Murine schistosomiasis: selective inhibition in vitro of fibroblast collagen production by mononuclear cells.

Primary cell cultures from the livers of mice infected with Schistosoma mansoni were prepared and cells with the appearance of fibroblasts by light microscopy were isolated. Collagen synthesis was estimated by measuring incorporation of 14C-proline into collagenase-sensitive proteins for both culture media and cell layers. Coculture of splenic T cells from infected mice with these hepatic fibroblasts caused greater selective and specific reduction in collagen production than did coculture using spleen cells from normal mice. There was a parallel inhibition in collagen within the cell layer which indicates that the marked decrease in collagen production was due to inhibition of synthesis and not related to changes in solubility or secretion. Primary culture of mouse skin fibroblasts showed similar responses to coculture but an established fibroblast line, 3T3, was unresponsive. Inflammatory cells appear to influence hepatic fibroblasts isolated under our experimental condition in several ways, such as opposite effects on collagen synthesis and cell proliferation.

Etiology of acute diarrhea among United States Embassy personnel and dependents in Cairo, Egypt.

The purpose of this study was to identify the enteropathogens causing acute diarrheal disease in Americans living in the North Africa/Middle East region during a 34-month period from February 12, 1985 to December 30, 1987 to guide preventive and therapeutic measures. Stool specimens were examined and an epidemiologic questionnaire was administered to patients with acute diarrhea at the Outpatient Health Unit of the United States Embassy in Cairo, Egypt. The subjects consisted of 126 American employees and dependents of the U. S. Embassy in Cairo, Egypt with diarrhea of less than two-weeks duration. Subjects received routine medical care administered by the U.S. Embassy Medical staff. A possible etiologic agent was detected in 41% of the subjects. Enteroadherent Escherichia coli was the most commonly isolated enteropathogen. A high degree of antimicrobial resistance was noted among the bacterial isolates, but all were susceptible to the quinolone antibiotics. Episodes of acute diarrhea occurring among American expatriates in Cairo, Egypt were primarily of bacterial etiology, but only a small portion were caused by the bacterial pathogens routinely identified in a standard clinical bacteriology laboratory. Most of the diarrheal episodes were due to noninvasive enteroadherent E. coli that may cause prolonged disease requiring antimicrobial therapy.

Hepatitis B and HIV in Sudan: a serosurvey for hepatitis B and human immunodeficiency virus antibodies among sexually active heterosexuals.

A serosurvey was conducted in Port Sudan and Suakin, Sudan in October and March 1987 to determine the prevalence and risk factors associated with the transmission of hepatitis B, human immunodeficiency virus type 1 (HIV-1), and syphilis among sexually active heterosexuals on the coast of Sudan. A total of 536 subjects, including 202 female prostitutes, 95 long-distance truck drivers, 103 soldiers, 72 Ethiopian refugees, and 54 Sudanese outpatients, were enrolled in the study. Seventy-eight percent (202/259) of the female study subjects were engaged in prostitution, and 57% (157/277) of the men admitted to prior sexual relations with prostitutes. Serologic markers for hepatitis B and syphilis were detected in 68% and 17% of the entire study population, respectively. In contrast, antibody to HIV-1 was detected in none of the 536 sera tested. Risk factors found to be independently predictive of hepatitis B infection by multivariate analysis included prostitution, positive serology for syphilis, and a history of anti-schistosomal therapy. The absence of HIV-1 infection among the prostitutes enrolled in this study is in marked contrast to the current AIDS epidemic in neighboring sub-Saharan countries, suggesting that HIV-1 has not been widely introduced on the coast of Sudan. The high prevalence of serologic markers to hepatitis B and syphilis, however, indicates a potential for HIV-1 in this region.

Low prevalence of HIV infection in Djibouti--has the AIDS epidemic come to a stop at the Horn of Africa?

To determine if the HIV-epidemic had reached Djibouti by autumn 1987, we investigated 645 subjects belonging to various risk groups; 150 were patients with a disease compatible with acquired immune deficiency or with a mycobacterial infection, 115 were young males having a sexually transmitted disease, 295 were female prostitutes, and 69 were villagers from a rural area; the remaining 16 belonged to other groups. All subjects answered an epidemiological questionnaire and had their serum tested for evidence of HIV antibodies. Eight sera were HIV-antibody positive by both ELISA and Western blot. Of these, 2 were from young men while 6 were from young women who admitted to prostitution. This accounts for an HIV seropositivity rate of 2.0% +/- 1.6% in the prostitute population. Also, one antibody-positive subject was positive for circulating HIV antigen. Seven of the seropositive individuals had no general complaints or abnormal clinical signs. The eighth subject was a 28 year old man in hospital for pneumonia. We conclude that in Djibouti, in late 1987, the prevalence of both AIDS and HIV infection in high risk individuals was much lower than that reported from other East African countries.

Viral hepatitis markers in Djibouti: an epidemiological survey.

A serosurvey involving 656 individuals revealed that hepatitis A infection was virtually universal in Djibouti in 1987, and more than half of the people investigated had serum markers of hepatitis B infection. The rate of chronic HBsAg carriers was 7.3% and was higher for males than for females. Both HBsAg and anti-HBs positivity rates were directly related to increasing age. No uniform mechanism could be found to account for transmission of the hepatitis B virus, and no correlation was noted between HBV-marker status and sexual promiscuity or the classic blood exposure risks. However, a significant association existed between the abuse of khat and the chronic HBsAg carrier state.

A study of viral and rickettsial exposure and causes of fever in Juba, southern Sudan.

Patients presenting at the Juba Teaching Hospital, either with fever of undetermined origin or with a clinical cause of fever, gave evidence of exposure to a wide range of viral and rickettsial agents. Serological tests showed high antibody levels to flaviviruses (56.9%) and alphaviruses (29.2%), with lesser levels of bunyamweraviruses (3.8%), Rift Valley fever (2.3%), and sandfly fever (0.75%). Flavivirus exposure was significantly associated with clinical evidence of liver disease; repeated exposure to flaviviruses was particularly prevalent in those with poor sanitation and who had received previous injections. A significant focus of Ebola and Marburg exposure in Juba has been identified. Clinical evidence of liver disease was evident in 37% of patients studied, and 24.6% were HBsAg positive. The first 2 HIV-positive individuals from the southern Sudan are reported, including one with clinical AIDS. A high prevalence of positive antibodies to Rickettsia typhi in the population indicated that murine typhus was common locally. This study indicates the need for further public health measures in the southern Sudan to control the spread of these infections.