Arterial oxygen saturation and diving response during dynamic apneas in breath-hold divers.

Competitive breath-hold divers try to achieve maximum times, depths, or distances underwater, thereby risking hypoxic syncope. In the present study, the cardiorespiratory responses to dynamic apnea (simultaneously initiated apneas and dynamic leg exercise) were investigated in 10 breath-hold divers. The divers performed 60 s dynamic apneas with the face in air (A) or face immersed in cold water (AFI). During apneas, the arterial oxygen saturation was reduced (A: -10%), but to a lesser extent during AFI (-6%, P<0.01), reaching a nadir 10-15 s post-apnea. Also, changes in end-tidal O(2) and CO(2) pressures (P(et)O(2)/P(et)CO(2)) were smaller during AFI than A (DeltaP(et)O(2): 8.2 vs 8.7 kPa, P<0.01; DeltaP(et)CO(2): 3.1 vs 3.2 kPa, P<0.05). The heart rate was lower during AFI than A (66 vs 78 bpm, P<0.01), reflecting an augmented diving response during AFI. The maximum safe breath-hold time under the conditions of the present study was calculated to be 101 and 106 s for A and AFI, respectively, consistent with the dynamic apnea times achieved by world-class apnea divers. It is concluded that the augmented diving response during face immersion apneas is associated with a slower reduction of the pulmonary (and arterial) oxygen store, probably delaying the occurrence of a hypoxic syncope.

Selected contribution: role of spleen emptying in prolonging apneas in humans.

This study addressed the interaction between short-term adaptation to apneas with face immersion and erythrocyte release from the spleen. Twenty healthy volunteers, including ten splenectomized subjects, participated. After prone rest, they performed five maximal-duration apneas with face immersion in 10 degrees C water, with 2-min intervals. Cardiorespiratory parameters and venous blood samples were collected. In subjects with spleens, hematocrit and hemoglobin concentration increased by 6.4% and 3.3%, respectively, over the serial apneas and returned to baseline 10 min after the series. A delay of the physiological breaking point of apnea, by 30.5% (17 s), was seen only in this group. These parameters did not change in the splenectomized group. Plasma protein concentration, preapneic alveolar PCO2, inspired lung volume, and diving bradycardia remained unchanged throughout the series in both groups. Serial apneas thus triggered the hematological changes that have been previously observed after long apneic diving shifts; they were rapidly reversed and did not occur in splenectomized subjects. This suggests that splenic contraction occurs in humans as a part of the diving response and may prolong repeated apneas.

Polymerase chain reaction for the early diagnosis of cytomegalovirus hepatitis in liver transplant patients.

BACKGROUND: In liver transplant (LTX) patients, cytomegalovirus (CMV) hepatitis as a cause of graft dysfunction occurs in 15-25% of the patients. Polymerase chain reaction (PCR), applied to liver biopsy specimens, may increase the ability to detect CMV DNA at a local site. In this study, PCR was used to compare its relation to the development of clinical CMV hepatitis. STUDY DESIGN: Nested polymerase chain reaction (nPCR), derived from a conserved region of the CMV major immediate-early gene, was used to examine 141 frozen liver biopsies from 61 LTX patients for the presence of CMV DNA. 134 biopsies were obtained from 54 patients with pathological liver function tests within four months after transplantation. The remaining seven patient biopsies were derived from the one-year investigation after LTX and served as controls. The results were compared to virus isolation, antigen detection by immunohistology and in situ hybridization for CMV DNA of the biopsy specimens. Histological examination was performed to verify a diagnosis of viral hepatitis. RESULTS: CMV DNA was amplified in 11% (15/134) of the biopsies, corresponding to 20% (11/54) of the patients. Virus isolation revealed CMV in 5% (7/134) of the samples. None of the nPCR-negative biopsies was virus culture positive. CMV genomes were detected by nPCR more frequently than CMV hepatitis was diagnosed by using the combination of CMV-specific histopathology and/or immunohistology and/or CMV-positive virus isolation (p < 0.01). However, when this comparison was performed within individual patients, the difference was not significant (p > 0.05). If the results of in situ hybridization were included in the diagnostic criteria of CMV hepatitis, the nPCR was comparable to these, both at the biopsy and the patient levels (p > 0.1 and p > 0.05, respectively). For the diagnosis of CMV hepatitis the negative predictive value of CMV-nPCR was 1.0. The positive predictive value ranged from 0.55 to 0.82 depending on the criteria of CMV hepatitis. The nPCR also detected signs of CMV infection in the liver graft earlier than virus isolation, 11 versus 21 days, respectively, after transplantation. CONCLUSION: The frequency of CMV DNA positivity, measured by nPCR, was similar to that revealed by other combined methods. We suggest that the combined findings of histological cholangitis and/or lobulitis together with nPCR for CMV DNA can be used as a diagnostic criterion for initiation of antiviral treatment against CMV hepatitis.

Effect of mycophenolate mofetil (RS-61443) on cytokine production: inhibition of superantigen-induced cytokines.

The effects of the immunosuppressant mycophenolate mofetil (MPAM, RS-61443) on cytokine production at the single cell level were assessed using in vitro activated human mononuclear cells. Cytokine production was studied with UV microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies (mAbs). The cytokines evaluated included interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10 interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), TNF-beta, and granulocyte macrophage-colony stimulating factor (GM-CSF). MPAM exhibited a marked antiproliferative effect without cytotoxicity in all mononuclear cell cultures. Six to 24 hours after stimulation with the superantigen Staphylococcus aureus enterotoxin A (SEA), most cytokine production was unaffected by MPAM at therapeutic concentrations (10(-6) M), with the exception of GM-CSF. In contrast, by 48 h after antigen activation, MPAM significantly inhibited all studied cytokine production (p < 0.05). Cyclosporin A (CsA), used as a control at a concentration of 100 ng/ml, inhibited production of all studied cytokines, at all time points. Monokine production after lipopolysaccharide (LPS) stimulation was unaffected by MPAM. Similarly, the production of most of the cytokines studied after mitogen stimulation with phorbol ester (PMA) plus calcium ionophore (ionomycin) was not affected by MPAM, in comparison to CsA which demonstrated significant inhibition of all cytokines tested under these conditions. However, a late inhibitory effect on IL-3 production was seen by MPAM at 48 h after mitogenic stimulation. Further observations are required to explain the divergent results on cytokine production by MPAM in superantigen-activated and mitogen-activated human mononuclear cells.

Down-regulation of cytokine production and interleukin-2 receptor expression by pooled human IgG.

The influence of pooled human IgG preparations for intravenous use (i.v.Ig) on in vitro-induced cytokine production was studied at the single-cell level using cytokine-specific monoclonal antibodies (mAb) and indirect immunofluorescent technique. Cultured mononuclear cells from peripheral blood from healthy adult donors were polyclonally stimulated for 96 hr by either direct ligation of T-cell receptors using immobilized anti-CD3 mAb or by a combination of a protein kinase C activator [phorbol 12-myristate 13-acetate (PMA)] and a calcium ionophore (ionomycin) in the absence or presence of i.v.Ig. A marked inhibition of proliferation and blast transformation was noted in all i.v.Ig exposed cultures, despite good cell survival. The production of the T-cell lymphokines interleukin-2 (IL-2), IL-10,interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) was significantly down-regulated during the whole studied period in the i.v.Ig containing anti-CD3 stimulated cultures. The synthesis of the monokine IL-8 was not suppressed and that of TNF-alpha, which was made by both lymphocytes and monocytes, was only moderately inhibited. Somewhat different and more transient effects were observed in the i.v.Ig-exposed PMA/ionomycin-activated cultures. The production of IL-2, IL-3, IL-4, IL-5, IL-10, TNF-beta and granulocyte-macrophage colony-stimulating factor (GM-CSF) was down-regulated during the initial phase of the cultures up to 48 hr, but not at 48-96 hr. The synthesis of IFN-gamma and TNF-alpha was unaffected of the influence of i.v.Ig during the entire culture period. The expression of IL-2 receptors (IL-2R) was significantly suppressed in the i.v.Ig-treated anti-CD3-activated cells, but not in the PMA/ionomycin-stimulated cultures. Taken together our results indicate that pooled IgG may mediate immunomodulation by direct effects on cytokine production and on T-cell proliferation.

Seroprevalence and outcome of hepatitis C in liver transplantation.

A recombinant enzyme-linked immunosorbent assay (ELISA) followed by a neutralization test (NT) and recombinant immunoblot assay (RIBA) were used for the detection of antibody to hepatitis C virus (anti-HCV) in 71 patients receiving 84 orthotopic liver grafts between 1984 and 1990. Before the liver transplantation (LTX) anti-HCV was present in six of the 71 recipients (8.5%) who were accepted for LTX because of acute or chronic liver failure. After LTX anti-HCV could not be detected in one of the patients, but it was continuously present in the others for more than 12 months. Detectable HCV antibodies were not present in the three patients who underwent LTX because of clinical evidence of fulminant NANB hepatitis. Two of 48 (4.2%) previously HCV seronegative recipients, who survived more than 3 months, seroconverted 9 and 16 months, respectively, after transplantation. The postoperative seroconversion was probably due to the transfer of virus via perioperative blood transfusions. Thus, these liver recipients may be able to respond by producing anti-HCV despite immunosuppressive therapy. None of the seven post-transplant HCV-seropositive patients developed symptoms such as icterus or fatigue, which would suggest the presence of liver insufficiency due to HCV infection. However, two of them had increased transaminase levels and histological signs of mild hepatitis. No significant difference was found in 1-year survival, prothrombin complex, albumin levels or the risk for retransplantation in post-transplant anti-HCV-seropositive patients, compared with those without detectable HCV antibodies (71% vs 69%, respectively). Thus, during the study period of 1-5 years, the clinical course of HCV infection was milder than that reported for hepatitis B infection in liver recipients.

Rapid detection of cytomegalovirus DNA and RNA in blood of renal transplant patients by in vitro enzymatic amplification.

Cytomegalovirus infection causes significant morbidity and mortality in renal transplant patients. The only marker of CMV infection that appears to correlate with the development of symptomatic illness is viremia. However, CMV grows slowly in tissue culture, requiring 2-6 weeks of incubation for detection of characteristic cytopathic effect. The efficacy of antiviral therapy for CMV may be improved by earlier detection of viremia and institution of antiviral therapy. We performed amplification of CMV DNA and RNA from peripheral blood of renal transplant patients using the polymerase chain reaction (PCR) technique. We consistently detected CMV DNA by PCR earlier than CMV was detected by culture. Detection of CMV RNA in one patient confirmed the presence of actively replicating virus in peripheral blood. Amplification of peripheral blood from healthy CMV-seropositive and seronegative individuals, and from seropositive renal transplant patients without evidence of active CMV disease, was consistently negative. These preliminary data indicate that PCR may provide a means for earlier diagnosis of CMV viremia. Future prospective studies should determine if early detection of CMV DNA by PCR in peripheral blood does predict viremia and symptomatic illness, and if earlier institution of antiviral therapy based on PCR results improves outcome for the CMV-infected transplanted patient.

Clinical aspects on Epstein-Barr virus infection.

Epstein-Barr virus infection (EBV) was discovered 25 years ago in tumour cells from Burkitt's lymphoma. Extensive virological studies have relieved that EBV causes infectious mononucleosis and contributes to the pathogenesis of Burkitt's lymphoma and nasopharyngeal cancer. Atypical courses of the primary infection may induce meningoencephalitis or hepatitis and are attracting increasing attention. Antiviral treatment with acyclovir has been administered for 7 days, intravenously or orally, in the early stages of infectious mononucleosis, in 2 placebo controlled trials. An inhibition of oropharyngeal EBV replication was verified but minimal effects on clinical symptoms was observed. A combination of intravenous acyclovir and prednisolone treatment for 10 days was therefore tried in 15 patients with fulminant mononucleosis in a pilot study. A transient cessation of virus shedding was noticed in all patients, and a substantial clinical effect on pharyngeal symptoms and on fever was seen in 12/15 patients within 3 days. Treatment with chemotherapy or irradiation is recommended in EBV-associated B-cell lymphomas seen in immunosuppressed, transplanted, or human immunodeficiency virus-seropositive patients. No effect of acyclovir has been reported, but such therapy may be considered in the early stage when EBV induces a polyclonal B cell activation. Acyclovir treatment is effective in the EBV-genome positive hairy leukoplakia noticed in human immunodeficiency virus-seropositive patients. However, no effect of any antiviral therapy has been reported in the X-linked lymphoproliferative syndrome affecting in particular 2-7 year old boys. Prophylactic use of immunoglobulin or acyclovir has been suggested in susceptible children. These results indicate that the variety of clinical manifestations induced by EBV at least partly depend on the immune response elicited in the host and not of virus replication per se. Therefore, treatment of these various disorders cannot be generalized but must be based on the use of antiviral drugs combined with immunomodulatory agents.

Human intravenous immunoglobulin modulates monokine production in vitro.

The effects of human immunoglobulin preparations for intravenous use (IVIg) on in vitro-induced monokine production were studied. Individual peripheral blood monocytes, obtained from healthy blood donors, which produced interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) after in vitro stimulation, were identified by cytokine-specific monoclonal antibodies (mAb) and indirect immunofluorescence technique. Lipopylosaccharide (LPS) or Borrelia burgdorferi spirochetes (Bb) were used to induce TNF-alpha and IL-6 production in cultures. Peak synthesis occurred 2.5 hr after initiation of the cultures in the majority of the monocytes, but not at all in lymphocytes. The monocytes were identified by two-colour staining using a monocyte-specific mAb. IL-6 was produced by 64 +/- 8% or 71 +/- 9% (means +/- SD) of the non-IVIg-exposed monocytes after LPS or Bb stimulation, respectively (n = 12). A dose-dependent and significant reduction of the number of IL-6-producing cells was noted in the IVIg-supplemented cultures (P less than 0.003). In these cultures 24 +/- 12% or 29 +/- 12% of the monocytes made IL-6 in response to LPS or Bb. Kinetic studies indicated a sustained significant inhibition of IL-6 production during 24 hr of culture (P less than 0.001). In contrast, TNF-alpha synthesis was not inhibited by IVIg. LPS or Bb stimulation resulted in 47 +/- 18% or 69 +/- 7% TNF-alpha producing cells versus 48 +/- 9% or 59 +/- 8% in IVIg-supplemented cultures. These results indicate down-regulation of IL-6, but not TNF-alpha production, by IVIg. A direct antigen neutralization is an unlikely explanation for the divergent effects observed on monokine production after IVIg addition.

Defective cell-mediated response to EBV-transformed B cells in a healthy individual with regular EBV antibody titers.

We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) who has regular antibody titers but defective inhibitory capacity toward the growth of autologous EBV-infected B cells. This in vitro function reflects the EBV-specific memory because it does not occur in experiments performed with cells of seronegative individuals. An analysis of events following in vitro EBV infection showed that lymphocytes of ST behaved in some tests in the same way as those collected from seronegative individuals. These parameters were: lack of gamma-IFN production 24 hr after EBV infection; low production of soluble factors that inhibit EBV-induced B-cell proliferation; lack of generation of LCL selective cytotoxicity after repeated stimulation with autologous LCL; and high proportion of EBNA-positive cells in 7-day-old EBV-infected cultures. On the other hand, cellular memory to the virus detected by the production of IL-2 24 hr after infection, and by the production of LIF upon exposure to EBV-encoded antigens, conformed with the results obtained with seropositive individuals. T-cell-mediated inhibition of EBV-induced B-cell growth in vitro has been regarded as a corollary of in vivo control of EBV-infected B cells. However, it is absent or has a low efficiency in certain disease categories which are not accompanied by risk of B-EBV growth. Our results with a healthy individual also indicate that several mechanisms contribute to a harmless life-long virus carrier state.

Cyclosporin A inhibits induction but not production of gamma interferon induced by Epstein-Barr virus.

Cyclosporin A (CsA) interferes with various T-cell functions in vitro and is a potent inhibitor of T-cell-dependent reactions in vivo, such as graft rejection and control of virus infections. Since human gamma interferon (Hu IFN-gamma) is synthesized by T cells and has a controlling role in regulation of Epstein-Barr virus (EBV) infection, we have studied the effects of CsA on EBV-induced cellular Hu IFN-gamma release. CsA inhibited dose-dependently the EBV-induced Hu IFN-gamma response, studied at the cellular level in human blood lymphocytes. These effects were not due to toxicity of CsA, since at inhibitory levels cellular EBV infection measured as polyclonal IgM production proceeded unaffected. CsA did not affect the number of spontaneous Hu IFN-gamma-secreting cells, nor did it have any inhibitory effect if added after virus exposure. It is concluded that CsA inhibits induction but not production of cellular Hu IFN-gamma.

Effects of pure interferons on Epstein-Barr virus infection in vitro.

Pure human gamma-interferon as well as alpha-interferon inhibited induction of immunoglobulin synthesis by Epstein-Barr virus but not by pokeweed mitogen in B lymphocytes from adult but not from newborn humans. The interferons inhibited the infected B lymphocytes directly, irrespective of the Epstein-Barr virus immune status of the donor, and their inhibitory effect was synergistic.