Originalia

~

A. Biglino, A. Surbone, F. Lipani, N. Cappello, B. Forno, A. M. Pollono, M. Busso, A. Pugliese

Spontaneous Release of Interferon as a Predictor of Clinical Evolution in HIV-Positive Subjects Summary: In order to establish a correlation with disease progression we prospectively evaluated ten clinical and immunologic parameters in 102 consecutive HIV-positive subjects. The eight immunologic variables were: in vitro spontaneous interferon release by peripheral blood monocytic cells, alpha- and gamma-interferon production induced by Newcastle Disease Virus and PHA, Multitest Mdrieux score, PHA- and CON-Ainduced lymphocyte transformation, absolute number of CD4 + cells and CD4/CD8 ratio, respectively. The two baseline clinical variables were risk factor and disease presentation. Generalized Wilcoxon analysis indi-

cated a significant correlation of one clinical (disease presentation at entry) and three immunologic variables (spontaneous interferon release, CD4 + cell number, Multitest M6rieux) with disease progression. Baseline spontaneous release of alpha, acid-labile interferon showed the best correlation with disease progression, and appeared to be significantly associated with CD4 + cell loss. Spontaneous release of acid-labile alpha interferon by mononuclear cells in vitro could be highly predictive of disease evolution in HIV-Ab positive, AIDSfree subjects.

Zusammenfassung: Spontane Interferon-Freisetzung als priidiktiver Parameter far den klinischen Ferlauf bei HIVpositiven Personen. Bei 102 konsekutiven HIV-positiven Personen wurden prospektiv zehn klinische und immunologische Parameter bestimmt und ihre Korrelation mit dem Krankheitsverlauf untersucht. Spontane Interferon-Freisetzung aus mononukle~iren Zellen des peripheren Blutes, durch Newcastle Disease Virus und PHA stimulierte Freisetzung von Interferon-alpha und -gamma, Multitest M6rieux-Score, PHA- und CONA-induzierte Lymphozytentransformation, absolute CD4+ Zellzahlen und CD4/CD8-Quotient waren die acht immunologischen Variablen, die in vitro bestimmt wurden. Die beiden klinischen Basisparameter waren Risikofaktoren und klinischer Zustand. Nur Feststel-

lung einer signifikanten Beziehung zwischen einem klinischen Parameter (klinischer Befund bei Aufnahme) und drei immunologischen Variablen (spontane Interferon-Freisetzung, CD4 +-Zellzahl und Multitest M6rieux) mit dem Fortschreiten der Krankheit wurde der Wilcoxon-Test herangezogen. Die eindeutigste Beziehung mit einem progressiven Krankheitsverlauf ergab sich ffir die spontane Freisetzung von s~iurelabilem Interferon; zugleich war dieser Parameter signifikant assoziiert mit einer Verringerung der CD4 + -Zellzahlen. Bei HIV-Antik6rper-positiven Personen, die noch keine Symptome von AIDS entwickelt haben, besitzt diese spontane Freisetzung von sfiurelabilem alpha-Interferon aus mononukle/iren Zellen in vitro mOglicherweise hohe pr~idiktiven Wert ftir den KrankheitsverlauL

Introduction Full-blown acquired immunodeficiency syndrome (AIDS) is characterized by marked alterations of cell-mediated immunity, both quantitative - such as decreased CD4 + cell number and reduced CD4 +/CD8 + cell ratio [1, 2] and functional, such as reduced lymphocyte transformation in response to mitogens and antigens [3-5], defective delayed-type hypersensitivity response [6], and impaired interferon and interleukin-2 (IL-2) production by mononuclear cells in vitro [7-9]. However, in HIV infection without evidence of AIDS, functional derangements of the immune response do not always reflect clinical deterioration and seldom provide predictive information about clinical evolution, with the exception of proliferative response to pokeweed mitogen [10]. Further search for better prognostic parameters is therefore warranted. Recently, spontaneous release of cytokines, and particularly of alpha-interferon has been observed in HIV-infected cell systems in vitro [11-12]. This finding supports previous observations on the presence of circulating alpha, acid-labile interferon

in patients with HIV infection [13]. In some studies [14], but not in others [15], this peculiar interferon has been considered as a prognostic parameter of clinical evolution. Spontaneous release of cytokines by peripheral blood mononuclear cells (PBMC) in vitro could be a sensitive marker of immunocompetent cell infection by HIV of other viruses, and thus a more reliable prognostic marker. For these reasons, we prospectively evaluated a cohort of 102 HIV-Ab positive, AIDS-free subjects, in order to establish ~ a correlation between spontaneous alpha-interferon release by PBMC and disease progression, and to elucidate any possible interdependence between this parameter and Received: 4 October 1990/Accepted: 31 October 1990 Dr.A. Biglino,Dr. F. Lipani, Dr. 13.Forno, Dr.A.M. Pollono,Dr. M. Busso, Prof.A. Pugliese, Istituto di Malattie Infettive,Universit~di Torino, Corso Svizzera 164, 1-10149 Torino; Dr. A. Surbone, Istituto Nazionale Tumori, Via Venezian 1, Milano;Dr. N. Cappello, Istituto di Genetica Medica, Universitadi Torino, Via Santena, 1-10149 Torino, Italy. This workwas supportedby a grant fromthe ItalianMinistryfor Education (Quota 60% - 1988/89).

Infection 19 (1991) No. 1 © MMV MedizinVerlag GmbH Mtinchen,MiJnchen1991

7 / 11

A. Biglino et al.: Interferon Release in HIV Infection other indicators of i m m u n e deficiency, such as clinical situations, CD4 + cell number, cutaneous delayed-type hypersensitivity, proliferative response to mitogens, alphaand g a m m a - i n t e r f e r o n production.

Cumulative % of patients with stable disease 1.2

D

1.0 0.8

Patients and M e t h o d s 0.6

Patients: 102 consecutive patients entered the study over a sixmonth period. Admission criteria included: positive immunoenzyme assay for antibodies to HIV-1 performed with two different commercial kits and confirmed by Western blot (DU PONT), and disease presentation in accordance with groups II (asymptomatic infection), III (persistent generalized lymphadenopathy or PGL) and IV-A, IV-B or IV-C2 (AIDS-related complex or ARC) as defined by the Centers for Disease Control (CDC) classification [16]. Exclusion criteria were: previous or ongoing antiviral or antineoplastic treatment; age below 18 or above 50 years; evidence of full-blown AIDS, as defined by the CDC classification [16]. Patients were followed every three months in order to detect clinical deterioration (i. e. clinical evidence of ARC or AIDS in patients with baseline asymptomatic disease or PGL, and manifestations of full-blown AIDS in patients with a baseline picture of ARC). Serum levels of HIV-Ag (enzyme immunoassay) as well as of anti-CMV and anti-EBV (VCA) IgG (indirect immunofluorescence) were determined at each control. Baseline parameters: The eight immunologic parameters considered at entry were: spontaneous Ifn release by PBMC in vitro; alpha- and gamma-Ifn production; concanavalin-A (CON-A)and phytohemagglutinin (PHA)-induced lymphocyte transformation; absolute number of circulating C D 4 + cells; C D 4 + / CD8 + cell ratio; Multitest Mdrieux score. The two baseline clinical variables were: disease presentation as defined by the already mentioned CDC classification system, and risk factors (i. v. drug addiction, homosexuality, polytransfusions, other factors). Cut-off values for interferon titres were chosen based on our laboratory standards obtained from a sample of 45 healthy subjects (4 + 3 I.U. for spontaneously released interferon, 512 + 384 I.U. for alpha-interferon production, and 128 + 64 I.U. for gammainterferon production; geometrical means with 95% confidence interval). Interferon evaluation techniques: Alpha-interferon was induced by stimulating 1 x 106 PBMC, obtained as described elsewhere [18], with Newcastle Disease Virus strain F (NDVF), at a multiplicity of 100:1 for 24 h. Gamma-interferon was induced by stimulating PBMC (1 x 106/ml) with PHA (Wellcome) at a final concentration of 1:40 for 72 h. Spontaneous interferon release was assessed on supernatants from PBMC cultures carried out in plain medium for 72 h. Interferon levels were evaluated on serial twofold dilutions of supernatants by a cytopathogenic effect reduction method [19], employing WISH human cells as a target, and vesicular stomatitis virus at a ratio of 1:1 as a challenge. Assays were performed on untreated supernatants, as well as after a 4 h incubation period at pH 2, and after a 2 h incubation with mouse anti-human alpha-interferon monoclonal antibody (MoAb) (Boehringer Mannheim). Interferon titres were expressed as international units (1 I.U. = the highest dilution protecting 50% of cell layer from VSV cytopathogenic effect). A working standard of human gamma-interferon was included in each assay. Correlations between spontaneous interferon release, clinical or serological evidence of other viral infections (HSV, VZV, CMV, EBV) and the presence of circulating HIVAg were investigated. Lymphocyte transformation techniques': PBMC at a concentration

12 / 8

0.4

0.2

Wilcoxon = 84.74

~-

P < 0.001

~

/

-+

0.0 10

20 Time (months) -o-

<81-U.--~

30

40

>=81.U.

Figure 1 : Kaplan-Meier plot of the proportion without disease progression of 102 patients on the basis of spontaneous interferon release (cut-off: 8 I.U.). of 1 x 106/ml were stimulated for 72 h in triplicate, in 96-wetl, flat-bottom microplates (Nunc) with PHA (Wellcome) and concanavalin-A (CON-A, Sigma), respectively, at a final concentration of 1:80 and 12.5 ~tg/ml, or with plain medium. 18 h before terminating cultures, 1 ~tCi/well of 3H-thymidine (Sorin) was added, cultures were then transferred on fiberglass paper with a semiautomated device (Titertek), and nuclide incorporation evaluated on a liquid scintillation system (LKB-Wallac). Lymphocyte transformation was expressed as the mean CPM difference between stimulated and control cultures. Cutaneous delayed hypersensitivity evaluation: All patients underwent a multi-puncture intradermal test (Multitest C.M.I., Mdrieux) including seven recall antigens (PPD, tetanus and diphtheria toxoids, Candida, Trichophyton, Proteus and Streptococcus antigens) and a glycerol control. Results were expressed as a score obtained by the sum (in mm) of the mean infiltration diametres after 48 h [20].

Peripheral T-cell characterization: Heparinized peripheral blood was analyzed with a Spectrum III (Ortho Diagnostics) device, employing OKT3, OKT4 and OKT8 monoclonal antibodies (Ortho).

Statistical analysis: Univariate survival analyses were based on the Kaplan-Meier product limit estimates of survival distribution [21]. Differences between survival curves were assessed using the generalized Wilcoxon test [22]. The relative importance of multiple prognostic factors on survival was estimated using the Cox proportional hazards regression model [23]. Linear correlation between parameters was assessed with Spearman's rank-correlation coefficient. All data were processed with BMDP statistical software produced by Health Science Computing Facility, UCLA [24].

Results Patients were followed for a m e d i a n of 32 m o n t h s (mean: 34.8; SD + - 10.5); 22 (21.6%) of t h e m showed progression of disease during follow-up. Baseline parameters as well as the influence of the considered variables on disease progression are r e p o r t e d in Table 1.

Infection 19 (1991) No. 1 © MMV Medizin Verlag GmbH Miinchen, Mfinchen 1991

A. Biglino et al.: Interferon Release in HIV Infection

1.2

Cumulative % of patients with stable disease

t.2 1.0

1.0 I

l

0.8

0.8 0.6

[m

0.6

[ 0.4

0.4 Wilcoxon

0.2

Cumulative % of patients with stable disease

Wilcoxon

= 15.88

P < O,OOl

0.0

P < 0,01 I

0

= 7.2

0.2

10

0.0

t

20 Time (months)

30

40

10

0

< = 400 --4- >400 Figure 2: Kaplan-Meier plot of the proportion without disease progression of 102 patients on the basis of CD4 + cell number.

Lymphocyte Transformation Tests The relation of disease progression to different levels of mitogen-induced lymphocyte transformation is reported in Table 1. Cumulative proportion of stable disease was low-

40

Figure 3: Kaplan-Meier plot of the proportion without disease progression of 102 patients on the basis of Multitest score. Cumulative % of patients with stable disease

Spontaneous and Induced Interferon Release The cumulative proportion of stable disease was greatly reduced in the 22 patients with significant levels (_> 8 I.U.) of spontaneously released interferon in vitro, if compared to the 80 patients whose mononuclear cells released no interferon (Wilcoxon test = 84.74; p < 0.0001) (Figure 1). Spontaneously released interferon was totally inactivated after treatment at pH 2, and after incubation with anti-human alpha-interferon MoAb. In five subjects, however, anti-alpha-interferon MoAb achieved only partial inactivation (50%), while the same amount of inactivation was obtained by incubation with anti-gamma-interferon MoAb, suggesting the possibility of a spontaneous gamma-interferon release, at least in some cases. Baseline spontaneous interferon release showed no correlation with the presence of HIV antigenemia, of mucocutaneous herpetic manifestations (caused either by VZV or HSV), or the level of anti-EBV (VCA) antibodies at any time during follow-up, and a weak positive linear correlation with antiCMV IgG level (r = 0.23). By contrast, a stronger positive linear correlation was evidenced between the amount of anti-CMV IgG titre increase (when observed during follow-up) and spontaneous interferon titre at the end of follow-up (r = 0.62). Reduced levels of PHA-induced gamma-interferon (< 64 I. U.) and of NDV-induced alpha interferon (< 128 I.U.) were observed in 32 and 64 subjects respectively. The cumulative proportion of stable disease did not differ significantly between patients with low versus normal production of both types of interferon (Table 1) (Wilcoxon test = 0.826 and 0.096, respectively; p = n.s. in both cases).

20 30 Time (months) S c o r e < l O - + - S c o r e > = 10

1.0

t

0.8

I

I

~

0.6

i

4--

i

L--~.~

~

.7.

0.4 Wilcoxon

= 8.96

0.2 P = 0.011

0.0

I

i

0

20 Time (months) ASYMPT. - 4 - L.A.S.

I

30

10

+

40

A.R.C.

Figure 4: Kaplan-Meier plot of the proportion without disease progression of 102 patients on the basis of clinical picture at entry.

er in patients with poor response to PHA, but showed no changes according to different levels of response to CON-A. These differences, though suggesting a connection between poor transformation to PHA and disease evolution, were not significant (Wilcoxon test = 12.824 for PHA and = 4.156 for CON-A; p = n.s. in both cases).

Peripheral T-cell Subsets and Multitest Response A significant correlation between disease evolution and both CD4 + cell number and MT score was observed, with a lower proportion of stable disease in patients with less than 400 CD4 + cells/Ixl (Figure 2; Wilcoxon = 15.880; p = 0.0009) and an MT score < 10 (Figure 3: Wilcoxon = 7.195; p = 0.0073). By contrast, no significant difference emerged from actuarial analysis concerning patients with different levels of CD4/CD8 ratio (Wilcoxon = 1.560; p =

0.4585) (Table 1).

Infection 19 (1991) No. 1 © MMV Medizin Verlag GmbH Mtinchen, Miinchen 1991

9 / 13

A. Biglino et al.: Interferon Release in HIV Infection

Table 1: Disease evolution according to clinical and immunologic parameters at entry. Results of generalized Wilcoxon test are reported.

Table 2: Multivariate analysis on all parameters considered at entry. Summary of stepwise results.

?il

64 38

15 7

23.4 18.4

0,826

0.3633

32 70

6 16

18.7 22.8

0.096

0.7569

80 22

3 19

3.7 86,3

84.743

0.0001

Gamma-interferon < >

64 = 64

®

Spontaneous interferon release CD4+/gl

Interferon levels (I.U.) Alpha-interferon < 128 > = 128

N

-

i!I~NN®NNN 0.5370

0.0852

34.714

< 0.001

0.0012

0.0007

3.425

0.060

Table 3: Correlation between spontaneous Ifn release and CD4 + cell loss (mean values with 95% confidence interval).

Spont. interferon < 8 > = 8

Mitogen response (D-CPM x 103) PHA < =

10 10-20 20-30 > 30 CON-A < = 10 10-20 20-30 > 30 C D 4 + (cells/}xl) < = 400 400- 800 800-1200 > 1200

20 23 28 31

5 6 7 4

25.0 26.0 25,0 12.9

12.824

45 28 18 11

13 3 4 2

28.8 10.7 22.2 18.1

4,156

15 55 20 12

8 9 2 3

53.3 16.3 10.0 25.0

30 50 22

8 11 3

26.6 22.0 13.6

10 = 10

34 68

13 9

Risk factor Homosexuals Drug addicts Others

24 62 16

7 11 4

15.88

0.0765

0.2451

0.0009

CD4/CD8 ratio < = 0.5 0.5-1.0 > 1.0

1.560

0.4585

38.2 13.2

7.195

0.0073

29.1 17.7 25.0

4.842

0.3039

Multitest score < >

Symptoms Asymptomatic P.G.L A.R.C.

40 50 12

10 7 5

25.0 14.0 41.6

8.958

0.0113

Clinical Parameters

The cumulative proportion of stable disease was lower in patients with a clinical picture of ARC or asymptomatic disease at entry if compared to the proportion observed in patients with a clinical picture of P.G.L. (Wilcoxon = 8.958; p = 0.0113) (Figure 4). By contrast, disease evolution was totally unaffected by risk factor (Wilcoxon = 4.842; p = n.s.) (Table 1). Finally, in order to select the most significant among the considered parameters, a multivariate analysis was performed. Two parameters (a number o f C D 4 + cells < 14 / 10

400/~tl and spontaneous release of alpha, acid-labile interferon) were selected by stepwise analysis (Table 2), suggesting a significant correlation with clinical deterioration (particularly strong for spontaneous interferon release). Correlations between Immunologic Parameters

No linear correlation was found between functional immunological parameters. A weak negative linear correlation (r = - 0.204; p = n.s.) was evidenced between the titre of spontaneously released interferon and CD4 + cell number at entry; indeed, patients with spontaneous interferon release showed a significantly stronger CD4 + cell loss at the end of follow-up than patients without interferon release (Table 3). Discussion Conflicting results have been reported in previsous studies concerning the correlation between abnormal functional parameters of cell-mediated immune response and clinical stage or disease evolution in HIV-infected subjects without evidence of AIDS. In this longitudinal study, based on a cohort of 102 HIVinfected, AIDS-free patients, we have demonstrated that lymphocyte proliferation to T-cell mitogens possesses a weak, though non-significant, correlation with disease evolution, while no correlation could be found between evolution and production of alpha- or gamma-interferon. Furthermore, these functional parameters appeared to be independent of quantitative assessment of circulating T-cell subsets. Among the other parameters considered at entry, three (spontaneous in vitro release of alpha, acid-labile interferon from PBMC, low number of circulating CD4 + cells, and a Multitest score of <10) were' significantly corelated

Infection 19 (!991) No. 1

© MMV Medizin Verlag GmbH MOnchen, Mt~nchen 1991

A. Biglino et al.: Interferon Release in HIV Infection

with further clinical deterioration. A negative linear correlation was found between spontaneous interferon titre and CD4 + cell number; consequently, a negative influence by spontaneously released alpha-interferon on peripheral T-cell number could be hypothesized. Recent studies evidenced cytokine release from cellular systems infected by retroviruses. In fact, HIV-infected CD4 + cells can induce the release of acid-labile alpha-interferon from B lymphocytes [11]. In addition, it has been shown that an acidstable alpha-interferon is spontaneously released by lymphnode leukocytes of lentivirus-infected sheep, and that this phenomenon is enhanced in the presence of lentivirus-infected accessory cells [12]. In our study, spontaneous alpha-interferon release was correlated with serological evidence of CMV infection, but not with the detection of circulating HIVAg; however, HIV antigenemia seldom reflects the severity of infection [25], and a better correlation could be found with plasma HIV titre (work in progress). It could be hypothesized that coinfection with CMV in early stages of disease may induce the release of abnormal amounts of cytokines by non-T cells, both circulating (monocytes, B lymphocytes) as well as resident (macrophages and dendritic cells). It is well known that alpha-interferons can enhance both T- and non-T-(NK-)mediated killing of virus-infected cells [26], either directly (NK cells) or indirectly (CTL), by enhancing the expression of class I MHC antigens on target cells [27]. Abnormal release of alpha-interferons by circulating non-T cells and/or by lymphoid tissue accessory cells could activate cytotoxic activities against infected CD4+ lymphocytes, and recruitment

of uninfected cells into peripheral sites of infection, (dendritic cells, macrophages) respectively, providing an increased number of susceptible targets for virus replication. This hypothesis is in accordance with the observation that CD4 + cell loss in HIV infection is disproportionately high in comparison with the actual number of infected cells, so that alternative mechanisms of cell death (autoimmune?) have been postulated [3, 28, 29]. Alternatively, both immunological and clinical disturbances (such as the wasting syndrome) could be explained by an alpha-interferon mediated enhancement of tumor necrosis factor (TNF) cytotoxic activity [30]; indeed, spontaneous release of this cytokine by PBMC of patients with HIV infection has been recently reported [31]. It is, however, improbable that the antiviral activity we observed in culture supernatants was due to TNF itself, as it was abolished by treatment at pH2 and by anti-human interferon-alpha MoAb treatment. Finally, Spontaneous interferon release could be a more sensitive indicator of progression than the detection of circulating alpha-interferon, as serum inhibitors of interferon activity [32] may lead to false-negative results. In conclusion, baseline spontaneous release of acid-labile alpha-interferon in vitro could be useful in predicting disease progression in HIV-infected patients, particularly when considered together with CD4 + cell number and clinical picture. Further studies are needed in order to elucidate the cell type releasing acid-labile alpha-interferon in humans and to explore the connections between this phenomenon, coinfection with other viruses, and enhanced cytotoxicity against CD4 + infected cells.

References 1. Clifford Lane, H., Fauci, A. S.: Immunologic abnormalities in the acquired immunodeficiency syndrome. Ann. Rev. Microbiol. 3 (1985) 477-500. 2. Seligmann, M., Pinching, A. J., Rosen, F. S., Fahey, J. L.: Immunology of human immunodeficiency virus infection and the acquired immunodeficiencysyndrome. Ann. Intern. Med. 107 (1987) 234-242. 3. Spickett, G. P., Dalgleish, A. C.: Cellular immunology of HIV infection. Clin. Exp. Immunol. 71 (1988) 1-7. 4. Ballett, J.-J., Couderc, L.-J., Rabian-Herzog, C., Duval-Roy, C., Janier, M., Danon, F., Clauvei, J.-P., Seligmann, M.: Impaired T-lymphocyte-dependent immune responses to microbial antigens in patients with HIV-l-associated persistent generalized lymphadenopathy. AIDS 2 (1988) 291-297. 5. Sharma, B., Gnpta, S.: Antigen-specific primary cytotoxic T lymphocyte (CTL) responses in acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC). Clin. Exp. Immunol. 621 (1985) 296-303. 6. Bratt, G., von Krogh, G., Moberg, L., Karlsson, A., Putkonen, P. O., Biherfeid, G., Bottiger, M., Sandstrom, E.: Intradermal testing with multiple recall antigens for identification of cell-mediated immune deficiency in homosexual men. Clin. Immunol, Immunopathol. 41 (1986) 206-215. 7. Murray, H. W., Scavuzzo, D. A., Kelly, C. D., Rubin, B. Y., Roberts, R. B.: T4 + cell production of interferon gamma and the clinical spectrum of patients at risk for and with acquired immunodeficiency syndrome. Arch. Intern. Med. 148 (1988). 1613-1616. 8. Rossol, S., Voth, R., Lauhenstein, H. P., Miiller, W. E., Schriider, H. C., Meyer zum Biischenfelde, K. H., Hess, G.: Interferon production

in patients infected with HIV-1. J. Infect. Dis. 159 (1989) 815-821. 9. Borzy, M. S.: Interleukin 2 production and responsiveness in individuals with acquired immunodeficiencysyndrome and the generalized lymphadenopathy syndrome. Cell. Immunol. 104 (1987) 142-153. 10. Hoffmann, B., Lindharflt, B., Gerstoft, J., Petersen, C. S., Platz, P., Ryder, L. P., Odum, N., Dickmeiss, E., Nielsen, P. B., Ullman, S., Svejgaard, A.: Lymphocyte transformation response to pokeweed mitogcn as a predictive marker for development of AIDS and AIDS related symptoms in homosexual men with HIV antibodies. Br. Med. J. 295 (1987) 293-296. 11. Capobianchi, M. R., De Marco, F., Di Marco, P., Dianzani, F.: Acidlabile human interferon alpha production by peripheral blood mononuclear cells stimulated by HIV-infected cells. Arch. Virol. 99 (1988) %19. 12. Lairmore, M. D., Butera, S. T., Callahan, G. N., DeMartini, J. C.: Spontaneous interferon production by pulmonary leukocytes is associated with lentivirus-induced lymphoid interstitial pneumonia. J. Immunol. 140 (1988) 779-785. 13. DeStefano, E., Friedman, R. M., Frieflman-Kien, A. E., Goedert, J. J., Henrikson, D., Preble, O. T., Sonnabend, J. A., Vilcek, J.: Acidlabile human leukocyte interferon in homosexual men with Kaposi's sarcoma and lymphadenopathy. J. Infect. Dis. 146 (1982) 451-455. 14. Eyster, M. E., Goedert, J. J., Poon, M. C., Preble, O. T.: Acid-labile alpha interferon; a possible preclinical marker for the acquired immunodeficiency in hemophilia. N. Engl. J. Med. 309 (1983) 583-586. 15. Chachoua, A., Krigel, R., Lafleur, F., Ostreicher, R., Speer, M , Laubenstein, L, Wernz, J., Rubenstein, P., Zang, E., Friedman-Kien, A.: Prognostic factors and staging classification of patients with epi-

Infection 19 (1991) No. 1 © MMV Medizin Verlag GmbH Miinchen, Mtinchen 1991

11 / 15

A. Biglino et al.: Interferon Release in HIV Infection demic Kaposi's sarcoma. J. Clin. Oncol. 7 (1989) 774-780. 16. Centers for Disease Control: Classification system for HLTV III/ lymphadenopathy-associated virus infection. M.M.W.R. 35 (1986) 334-339. 17. Centers for Disease Control: Revision of the CDC Surveillance Case Definition for Acquired Immunodeficiency Syndrome. M.M.W.R. 36 (1987) (Supplement) 1-15. 18. B6ynm, A.: Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97) (1968) 77-89. 19. Biglino, A., Forno, B., Pollono, A. M., Busso, M., Maseolo, M., lhlgli. ese, A., Tovo, P., Gioannini, P.: Effect of erythromycin on the immune response and interferon production. Immunopharmacology 14 (1987) 101-106. 20. Kniker, W. T., Anderson, C. T., Roumiantzeff, M.: The multi-test system; a standardized approach to evaluation of delayed hypersensitivity and cell-mediated immunity. Ann. Allergy 43 (1979) 73-78. 21. Kaplan, E. L., Meier, P.: Non parametric estimation for incomplete observations. J. Am. Stat. Assoc. 53 (1958) 457-481. 22. Gehan, E.: A generalized Wilcoxon test for comparing arbitrarily singly-censored samples. Biometrika 52 (1965) 203-223. 23. Cox, D. IL: Regression models and life tables (with discussion). J. R. Stat. (Series B) 34 (1972) 187-220.

24. BMDP: Statistical Software. University of California Press, Berkeley 1981, pp. 576-583. 25. Coombs, R. W., Collier, A. C., Ailain, J.-P., Nikora, B., Leuther, M., Gjerset, G. F., Core)', L.: Plasma viremia in Human Immunodeficiency Virus infection. N. Engl. J. Med. 321 (1989) 1626-1631. 26. Pestka, S., Langer, A., Zoon, K. C., Samuel, C. E.: Interferons and their actions. Ann. Rev. Biochem. 56 (1987) 727-777. 27. Balk'will, F. R.: Interferons, Lancet i (1989) 1060-1063. 28. Edeiman, A. S., Zolla-Pazner, S.: AIDS: a syndrome of immune dysregulation, dysfunction, and deficiency. FASEB J. 3 (1989) 22-30. 29. Aseher, M. S, Sheppard, H. W.: AIDS as immune system activation: a model for pathogenesis. Clin. Exp. Immunol. 73 (1988) 165-167. 30. Lau, A. S., Livesey, J. F.: Endotoxin induction of tumor necrosis factor is enhanced by acid-labile interferon-alpha in acquired immunodeficiency syndrome. J. Clin. Invest. 84 (1989) 738-743. 31. Roux.Lombard, P., Modoux, C., Cruchaud, A., Dayer, J. M.: Purified blood monocytes from HIV 1-infected patients produce high levels of TNF alpha and IL-1. Clin. Immunol. Immunopathol. 50 (1989) 374-384. 32. Ikossi-O'Connor M. G., Chadha, K. C., Lillie, M. A., Bernstein, Z., Zueker-Franldin, D., Ambrus, J. L.: Interferon inactivator(s) in patients with AIDS and AIDS-unrelated Kaposi's sarcoma. Am. J. Med. 81 (1986) 783-785.

Book Review

N. C. Khan, J. L. Melnick (eds.) Human Immunodefieieney Virus: Innovative Techniques for Isolation and Identification Karger, Basel 1990 130 pages, 31 figures, 14 tables Price: SFr. 134,-, US-$ 99.25 This book contains seven different chapters dealing with the different methods for the detection of HIV. Chapter 1 gives a review of the isolation of HIV from infected tissue fluids, tissues and cells citing various references. Neither a recommendation for a successful procedure is given nor a scoring of the validity of the different methods. Chapter 2 describes the detection of HIV nucleic acid in clinical samples. Besides a recommended procedure and precautions necessary working with amplified materials, the configuration of a new sandwich hybridization assay in microti.ter plates is described for the handling of large numbers of specimens after PCR amplification. Specificity of this new assay is still a problem. Chapter 3 gives a summary of the CD4 lymphocyte numbers, neopterin and 132 microglobulin levels during the course of the HIV infection. Additionally to clinical markers these parameters can be used for a prognosis of the immunodeficiency and the onset of treatment in most of the patients followed permanently over some years. Chapter 4 deals with the diagnostic value of the determination of p17 antigen and anti-pl7 by using different peptides. The C-terminal part of this core protein may be a useful additional marker besides the p24 antigen when the problems with the sensitivity

16 / 12

and specificity of the assay have been resolved. In the fifth chapter a bioassay for the detection of HIV transactivating capacity is cited using different references. This assay may be valuable for the monitoring of drugs interfering in the replication cycle of HIV and for the characterization of isolated viruses from patients as rapid/high and slow/low variants. Chapter 6 describes the different methods for the diagnosis of HIV-1 and HIV-2 infection either by the detection of HIV specific antibodies or antigens. The cited assays are commercially available using recombinant antigens. The configuration of a new competitive monoclonal immunoassay for the distinction of HIV-1 and HIV-2 antibodies with recombinant transmembrane proteins is explained. Finally, Chapter 7 consists of a detailed description of HIV-1 isolation from peripheral blood lymphocytes in a microculture assay. The correlation of the patient's cell number and that of the acceptor cells has been evaluated and a definition of a positive culture result is also given. This book (130 pages) supplies a nice summary of the current literature of HIV isolation as well as the detection of HIV nucleic acids, antigen and antibodies. If the reader wants to see a detailed description of a procedure and the scoring of the efficacy and validity of the cited procedures, with the exception of Chapters 2 and 7, he will remain in the dark. Therefore the title of this booklet is misleading. Until the different cited techniques can be used in the laboratory a lot of library and laboratory work has to be done.

L. Gftrtler Miinchen

Infection 19 (1991) No. 1 © MMV Medizin Verlag GmbH MiJnchen, Miinchen 1991