Bo Hofmann

The Prognostic Value of Cellular and Serologic Markers in Infection with Human Immunodeficiency Virus Type 1

We evaluated three cellular and five serologic markers that are affected by infection with the human immunodeficiency virus type 1 (HIV-1) for their ability to predict the progression to clinical acquired immunodeficiency syndrome (AIDS). The cellular markers were the number of CD4+ T cells, the number of CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells. The serologic markers were the serum levels of neopterin (a product of stimulated macrophages), beta 2-microglobulin, soluble interleukin-2 receptors, IgA, and HIV p24 antigen. We evaluated the usefulness of these measures as markers of the progression to AIDS prospectively, over four years, in a cohort of 395 HIV-seropositive homosexual men who were initially free of AIDS. CD4+ T cells (expressed as an absolute number, a percentage of lymphocytes, or a ratio of CD4+ to CD8+ T cells) were the best single predictor of the progression to AIDS, but the serum neopterin and beta 2-microglobulin levels each had nearly as much predictive power. The neopterin level appeared to be a slightly better predictor than the beta 2-microglobulin level. The levels of IgA, interleukin-2 receptors, and p24 antigen had less predictive value. A stepwise multivariate analysis indicated that the best predictors, in descending order, were CD4+ T cells (the percentage of lymphocytes or the CD4+: CD8+ ratio), the serum level of neopterin or beta 2-microglobulin, the level of IgA, that of interleukin-2 receptors, and that of p24 antigen. The last three markers had little additional predictive power beyond that of the first two. We conclude that of the eight markers studied, progression to AIDS was predicted most accurately by the level of CD4+ T cells in combination with the serum level of either neopterin or beta 2-microglobulin. At least one of these two serum markers, which reflect immune activation, should be used along with measurement of CD4+ T cells in disease-classification schemes and in the evaluation of responses to therapy.

Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin

BACKGROUND Low serum concentrations of mannose-binding lectin (MBL) are associated with increased susceptibility to recurrent infection. Three variant alleles in the MBL gene (B, C, and D), cause low serum concentrations of the protein. We investigated whether variant alleles of MBL affect susceptibility to infection with HIV and progression of AIDS. METHODS Between 1983 and 1986, all men who attended two clinics in Copenhagen for HIV screening were invited to take part in our study. We investigated the prevalence of variant alleles of MBL (detected by PCR) and assessed the prognostic value of these alleles and the corresponding serum MBL concentrations (measured by ELISA) in 96 homosexual men with HIV infection and in two control groups (123 healthy adults and 36 HIV-negative homosexual men at high risk of HIV infection because of their sexual behaviour). Follow-up was for up to 10 years. FINDINGS Eight (8%) of the HIV-infected men were homozygous for the variant MBL alleles compared with one (0.8%) of the healthy controls (p = 0.005) and none of the high-risk homosexual controls (p = 0.05). We found no significant association between MBL genotype and time from first positive HIV test to progression of AIDS (p = 0.8). However, in the 61 HIV-infected men who developed AIDS, the median survival time was significantly shorter after the AIDS diagnosis for men who were carriers of the variant alleles (both homozygous and heterozygous) than for men homozygous for the normal MBL allele (11 [IQR 4-21] vs 18 months [9-44], p = 0.007). Among men who developed AIDS, there was a significant difference in survival time between those with serum MBL concentrations below the lower quartile, those within the IQR, and those above the upper quartile (p = 0.02). Multivariate analysis showed that men who developed AIDS and had low serum MBL concentrations had an increased rate of rapid death, independently of CD4 T-cell counts at AIDS diagnosis. INTERPRETATION Our findings suggest that homozygous carriers of variant MBL alleles are at increased risk of HIV infection, either directly or indirectly because of increased susceptibility to coinfections. These alleles are also associated with a significantly shorter survival time after a diagnosis of AIDS.

Increased frequency of homozygosity of abnormal mannan-binding-protein alleles in patients with suspected immunodeficiency

A low plasma concentration of mannan-binding protein (MBP) impairs opsonisation and phagocytosis. Three different mutations in the MBP gene have a dominant effect on MBP concentration. We investigated the frequency of the abnormal MBP alleles in 228 unrelated patients suspected of various non-HIV-related immunodeficiencies. The frequency of heterozygotes for the abnormal alleles was not different from that in the background population (36.0% and 37.4%, respectively). By contrast, the frequency of homozygotes for the abnormal alleles was significantly increased (8.3% and 0.8%, respectively; p = 0.0017). This finding implies that homozygotes for abnormal MBP alleles are predisposed to recurrent infections.

Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival and slower CD4 T-cell decline in a cohort of HIV-seropositive individuals.

Objective:Recently, it has been shown that a homozygous 32 base-pair deletion in the gene encoding CKR-5, a major coreceptor for HIV-1, leads to resistance to infection with HIV-1. We have investigated whether HIV-seropositive individuals who were heterozygous for the CKR-5 deletion had a different course of the disease. Design:Thirty-five high-risk HIV-seronegative and 99 HIV-seropositive Danish homosexual men followed from 1985 to 1996 and 37 blood donors were analysed for their CKR-5 genotype by polymerase chain reaction. Results:Two (6%) of the 35 HIV-seronegative subjects at high-risk of infection were homozygous and seven (20%) were heterozygous for the CKR-5 deletion. This was not significantly different from the distribution in normal donors. Twenty-two (22%) of the 99 HIV-seropositive subjects were heterozygous and none was homozygous. Two subgroups of patients who had an opposite course of the HIV disease were identified. Of nine long-term non-progressors, six (66%) were heterozygous for the deletion. This frequency is significantly higher than in nine rapid progressors of whom none was heterozygous. The frequency of heterozygotes in long-term nonprogressors was also significantly higher than in the cohort as a whole. A Kaplan-Meier plot of the HIV-seropositive subjects, of whom 57 developed AIDS, showed a significantly better prognosis within the first 7 years of follow-up for those who were heterozygous for the deletion. Heterozygous individuals also had a significantly slower decrease in CD4 T-cell count per year. Conclusion:Individuals who are heterozygous for the 32-base-pair deletion in the CKR-5 gene have a slower decrease in their CD4 T-cell count and a longer AIDS-free survival than individuals with the wild-type gene for up to 11 years of follow-up.

Development of cytomegalovirus (CMV) disease may be predicted in HIV-infected patients by CMV polymerase chain reaction and the antigenemia test

Objective:Cytomegalovirus (CMV) is a frequent opportunistic viral pathogen in patients with AIDS leading to retinitis and other serious manifestations. CMV disease may be successfully treated. Prophylactic antiviral therapy has been shown to reduce the risk of CMV disease if initiated early. We evaluated PCR and the antigenemia test as methods for early detection of CMV disease. Methods:Two-hundred HIV-seropositive subjects with CD4 T-cell counts below 100 × 106/l were monitored with CMV polymerase chain reaction (PCR), the antigenemia test, blood cultures and CMV immunoglobulin (Ig) G and IgM titres every second month for 1 year. Results:Thirty-eight patients (19%) developed CMV disease. The PCR test detected CMV DNA a median of 46 days before onset of disease. This was earlier than the median of 34 days for the antigenemia test and a median of 1 day for CMV blood cultures. Univariate analysis showed that the CMV PCR, the antigenemia test and blood cultures all had significant predictive values for subsequent development of CMV disease with odds ratios (OR) of 30, 22 and 20. CMV serology had no predictive value. Multivariate analysis showed that the PCR method was superior to the other tests; OR: CMV PCR 10.0, antigenemia test 4.4 and CMV cultures 4.3. No clinical parameters had any significant predictive value in the stepwise multivariate model. Conclusions:The CMV PCR and the CMV antigenemia tests are both sensitive methods that may predict development of CMV disease up to several months prior to clinical disease. These methods make it possible to select patients at high risk for CMV disease and suitable for prophylactic therapy against CMV.

Serum beta 2-microglobulin level increases in HIV infection: relation to seroconversion, CD4 T-cell fall and prognosis.

Beta2-microglobulin (β2-M), a marker that is increased in serum during immune activation, was investigated during the course of HIV infection. β2-M rose promptly in the first phase of HIV infection in people who were participating in a longitudinal study where serum samples and lymphocyte subset data were obtained at 6-monthly intervals. A rise in β2-M level in the first seropositive sample was seen in 93% of 50 HIV seroconverters, and those with high (or low) levels of β2-M at the end of year 1 tend to remain high (or low) in the ensuing years. Eighty-three per cent of seroconverters experienced a fall in CD4 T cells in the first year. The magnitude of the CD4 T-cell decline, however, did not correlate with the rise in β2-M in specific individuals in the first year. Nevertheless, 2–3 years after seroconversion, the initially increased β2-M levels did correlate inversely with the (reduced) level of CD4 T cells (P < 0.001). Thus, the pattern of disease reflected by β2-M level is established in the first year of infection and persists through the following 2 years. β2-M levels were found to correlate with rate of CD4 T-cell fall in individuals with established HIV infection. Three groups of HIV-seropositive people with similar CD4 T-cell numbers at the first measurements (about 600–800 × 106/l) but different rates of CD4 T-cell fall over the following 2 years were evaluated by β2-M levels. The group with stable CD4 T-cell numbers showed a significantly lower level of β2-M than the groups with moderately or rapidly declining CD4 T-cell numbers. Increases in β2-M levels during the 2 years of observation were found in people exhibiting a rapid decline in CD4 T cells (about 200 cells/year). The level of β2-M appears to be an indicator of HIV activity and of the rate of CD4 T-cell fall. Serum β2-M level was also found to be a good indicator of AIDS occurrence within 3 years. In a study of 399 men who were seropositive and without AIDS at entry, β2-M and CD4 T cells had about equal predictive power, but the combination of CD4 T cell and β2-M levels provided a more powerful prognostic ability than either alone.

Restoration of T-cell function in HIV infection by reduction of intracellular cAMP levels with adenosine analogues.

Objective and designThe impaired function of T cells is characteristic of HIV infection. It contributes to disease pathogenesis and is associated with disease prognosis. Our aim was to describe a biochemical basis for this impairment and a pharmacological way of restoring function. MethodsMeasurement of intracellular cAMP, protein kinase A (PKA) activity and proliferative capacity of T cells to recall antigens. ResultsHIV-seropositive individuals without AIDS showed significant increases in intracellular cAMP levels and PKA activity (inhibitors of lymphocyte function). The proliferative capacity of T cells to recall antigens correlated inversely with initial cAMP levels: poor proliferation was associated with high cAMP level in HIV infection. Moreover, drugs that reduced intracellular cAMP levels led to significant restoration of specific T-cell proliferation and cytotoxicity. ConclusionsOur findings indicate that high intracellular cAMP concentrations contribute to pathogenesis of T-cell anergy in HIV infection and that drugs that decrease intracellular cAMP levels may be beneficial in the treatment of AIDS.

HIV-infected individuals with the CCR delta32/CCR5 genotype have lower HIV RNA levels and higher CD4 cell counts in the early years of the infection than do patients with the wild type. Copenhagen AIDS Cohort Study Group.

The relations among serum HIV RNA levels, CD4 cell counts, presence of the mutant CCR5-allele in heterozygous form, and clinical outcome was analyzed in 96 patients from the Copenhagen AIDS Cohort. In the early years of the infection, patients with the CCR5 delta32/CCR5 genotype had significantly lower HIV RNA levels (p = 0.005) and higher CD4 cell counts (p < 0.005) than did patients homozygous for the normal allele. The long-term clinical benefit of being heterozygous is small and cannot solely explain the large interpatient variation in progression rates. The beneficial effect of being heterozygous seems to be mediated by events in the early stages of the HIV infection.

Serum increases and lymphoid cell surface losses of IL-2 receptor CD25 in HIV infection: Distinctive parameters of HIV-induced change

Lymphocyte activation induces production of soluble IL-2 receptor (sIL-2R) which is a large portion of the CD25 membrane molecule and which is detectable in serum. Serum sIL-2R is reported here to increase as a direct effect of the HIV infection and not to be due to secondary opportunistic infections. sIL-2R increased promptly after HIV seroconversion in 83% of 50 initially seronegative homosexual men. The sIL-2R serum levels stabilized in the third year after seroconversion and were then predictive of later CD4 T cell levels and development of AIDS. In two studies of 59 and 395 seropositive men, beta-2 microglobulin (B2M) and neopterin levels in serum correlated closely with each other but not with sIL-2R levels. Thus, increased production of sIL-2R may reflect pathological processes distinct from those determining B2M and neopterin increases. Membrane CD25 expression on peripheral blood lymphocytes, unexpectedly, was found to be decreased in HIV infection. This contrasted with the increased sIL-2R in serum. Investigations with sensitive flow cytometry technics showed that CD25 was expressed at reduced levels and averaged only 12% of lymphocytes from HIV-infected individuals in contrast to 25% in noninfected individuals. All major lymphoid populations showed reductions in CD25 positive cells. This reduction in lymphoid membrane CD25, however, was not inversely correlated with the increased serum levels of sIL-2R or with other parameters of immune deficiency or activation. Thus, surface CD25 loss and serum sIL-2R increase are separate and independent consequences of HIV infection.

Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relation to AIDS

The tumor-promoting sesquiterpene lactone, thapsigargin, induced a dose-dependent increase of the cytoplasmic Ca2+ concentration ([ Ca2+]i) in human lymphocytes from a resting level between 100 and 150 nM up to about 1 microM. Half-maximum response was found at about 1 nM of thapsigargin, full response at 100 nM. The effect of thapsigargin on [Ca2+]i exceeded that of phytohaemagglutinin (PHA) which raised [Ca2+]i to maximum 300 nM. In combination with phorbol 12-myristate 13-acetate (PMA), thapsigargin stimulated the proliferation of normal lymphocytes to the same extent as did PHA, whereas the thapsigargin/PMA treatment could not restore the defective proliferation of AIDS lymphocytes in spite of the increased [Ca2+]i. Thapsigargin or PMA added separately had no stimulatory effects on cell proliferation. The thapsigargin/PMA treatment caused an increase in the interleukin-2 (IL-2) production of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects on lymphocyte proliferation by increasing [Ca2+]i, and that the general defect of AIDS lymphocytes, rather than being ascribed to the initiating signal systems, is associated with later events related to DNA synthesis and proliferation.