Glucose-6-phosphate dehydrogenase deficiency neonatal screening: preliminary evidence that a high percentage of partially deficient female neonates are missed during routine screening.

OBJECTIVES: To provide preliminary evidence that the currently employed semiquantitative method of screening for glucose-6-phosphate dehydrogenase (G6PD) deficiency can only detect infants who are totally deficient for G6PD and misses all cases of partial G6PD deficiency. SETTING: General population: 2150 randomly selected blood samples from the Blood Donation Department, Speliopouleion General Hospital, Athens, Greece. Neonate population: 2000 samples from neonates (50% male; 50% female) in maternity hospitals in the greater Athens area. High risk population: a total of 545 individuals from 133 families in the Athens area, the minimum acceptance criteria being the parents and any brother or sister. METHOD: Blood specimens from neonates or adults were collected and either spotted and dried on special filter paper (Schleicher and Schull 2992, Darmstadt, Germany) or used in tubes after being heparinised. For the quantitative evaluation of G6PD enzyme activity, the Quantase G6PD screening kit (Quantase Limited, Perth, UK) was used. Quantase G6PD controls (Quantase Limited) were used at three levels of G6PD. These controls are rated at 24, 30, and 37 degrees C. Alternatively, we used the Sigma G6PDH controls (Sigma Chemical Company, St Louis, USA) which are rated at 30 and 37 degrees C. The assay was performed according to the instructions included in the kit with the modification for haemoglobin normalisation. RESULTS: General population: 36 females who were classified as having normal enzymatic activity with the semiquantitative test, were classified as partially deficient with the quantitative test. Neonate population: using the quantitative test, the percentage of G6PD deficient neonates in this population was 5.5%, compared with 3.17% reported in routine screening using the semiquantitative method. High risk population: the quantitative method detected 28 cases of total or partial G6PD deficiency in sisters of males with known total deficiency. The semiquantitative CONCLUSIONS: A considerable amount of partially G6PD deficient female neonates (heterozygotes) are undetected and classified as having normal enzymatic activity using the semiquantitative method, which uses a cut off of 2.1 U/g haemoglobin (Hb). The use of a fully quantitative G6PD screening kit is proposed, employing the automated haemoglobin normalisation and a cut off of 6.4 U/g Hb. Any neonate with an activity below this mark should be regarded as G6PD deficient, and all preventive measures should be taken.

Elevated prostaglandin E2 production by monocytes is responsible for the depressed levels of natural killer and lymphokine-activated killer cell function in patients with breast cancer.

BACKGROUND: Human natural killer (NK) cells mediate spontaneous cytotoxicity against tumor cells and represent the main precursors of lymphokine-activated killer (LAK) cell activity. A comparison of some aspects of NK and LAK cell activity was undertaken in 85 preoperative patients with breast cancer and 75 healthy donors. METHODS: NK cell activity (tested in 18-hour cultures of effector peripheral blood mononuclear cells [PBMC] with K562 or MOLT-4 tumor target cells) was significantly diminished in these patients as it was the fully mature LAK cell activity (i.e., interleukin-2 (IL-2)-induced cytotoxicity in PBMC) against NK resistant target cells. Using immunoenzymatic methods we showed that the reduced NK cell activity was due to abnormally high levels of prostaglandin E2 (PGE2) produced by monocytes in culture. RESULTS: PGE2 was found to suppress the production of IL-2 in these cultures. Removal of monocytes from PBMC restored to almost normal levels the deficient NK and LAK cell activity in patients with breast cancer and was also associated with a normalization in the levels of PGE2 and IL-2. Indomethacin and gamma-interferon (IFN-gamma) increased the NK and LAK cell activity in these patients up to the levels of healthy donors. When highly purified CD56+ cells (obtained by an immunomagnetic isolation technique) were used as effector cells, no differences in LAK cell activity could be noticed between healthy donors and patients with cancer. FACS and northern blot analyses demonstrated a PGE2-mediated down-regulation of IL-2 receptor (IL-2R) expression on CD56+ cells that correlated with reduced LAK cell activity. This inhibitory effect of PGE2 was noticeable in long-term LAK cultures and was abrogated in the presence of IFN-gamma or indomethacin. CONCLUSION: This study may have important implications in the potentiation of NK and LAK cell activity for immunotherapeutic protocols in patients with breast cancer.

Prothymosin alpha restores depressed allogeneic cell-mediated lympholysis and natural-killer-cell activity in patients with cancer.

Cancer-bearing patients exhibit a variety of profound T-cell abnormalities which include decreased cytotoxic capacity as measured by allogeneic cell-mediated lympholysis (CML), natural-killer (NK) cell activity, and decreased lymphokine production. In patients with advanced solid malignancies, allogeneic CML, tested by a 4-hr 51Cr-release assay, was significantly lower than in a group of normal individuals. If optimal doses of affinity-purified prothymosin alpha (ProT alpha) were present during mixed lymphocyte culture, the CML of cancer patients was increased almost to normal levels. Mixed lymphocyte reaction, tested by tritiated thymidine uptake, was also decreased in these patients and was enhanced to normal levels if ProT alpha was added to the cultures. NK activity was decreased in these patients according to 51Cr-release assays. ProT alpha increased the NK activity up to normal levels. The reduced NK and CML activities in cancer patients were associated with abnormal production of prostaglandin E2 (high) and interleukin-2 (low), which were to a great extent normalized in the presence of ProT alpha. These results demonstrate that ProT alpha is capable of potentiating or fully restoring the deficient cytotoxic effector function of peripheral mononuclear cells (MNC) in patients with advanced malignancies.

Comparison of immune parameters in patients with one or two primary malignant neoplasms.

The purpose of this study was to correlate cellular immune responses and cytokine production in vitro between patients with one or two primary malignant neoplasms. One hundred and ninety-three patients (110 patients with one primary malignant neoplasm (group I), and 83 patients with two primary tumors (group II), entered this study. Mononuclear cells isolated from peripheral blood were tested in the following tests: (a) proliferative responses in the autologous and allogeneic mixed lymphocyte reaction (auto- and allo-MLR respectively): (b) natural killer cell activity; (c) production of interleukin-2 during the allo-MLR, and (d) interleukin-1 beta production by lipopolysaccharide-stimulated monocytes. All these parameters were found to be decreased in cancer patients as compared to normal donors (p < 10(-3)). In addition, we were able to detect significant differences between the values obtained from patients in groups I and II (p < 10(-2)). These data suggest a further impairment in cancer patients' immune status after the diagnosis of a second malignancy.

Sister-chromatid exchange in highly purified human CD4+ and CD8+ lymphocytes.

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD4+ and CD8+ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD4+ lymphocytes showed significantly higher SCE frequencies than autologous CD8+ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD4+ and CD8+ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD4+ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD8+ lymphocytes. Abnormalities in CD4+ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.

Prothymosin alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation.

Prothymosin alpha (ProT alpha) is an acidic polypeptide with potentiating effects on HLA-DR-restricted in vitro cellular immune response systems such as T cell proliferative responses to soluble proteins and cellular auto- or alloantigens. Experiments were performed to investigate the effect of ProT alpha on MHC class II Ag expression in human monocytes, murine splenocytes, and tumor cell lines at both protein and molecular levels. RIA and immunofluorescence analysis revealed that ProT alpha enhances HLA-DR surface Ag expression whereas Northern blot analysis demonstrated that ProT alpha causes significant accumulation of MHC class II mRNA. The enhancing effect of ProT alpha was demonstrated convincingly using precultured human peripheral monocytes, which are known to express decreased amounts of surface HLA-DR Ag, and HLA-DR-positive human cell lines. Moreover, ProT alpha was shown to induce HLA-DR Ag expression in a priori HLA-DR-negative tumor cells. Furthermore, ProT alpha was shown to be active in vivo. Splenocytes from mice pretreated with ProT alpha expressed more surface Ilpha Ag and contained more I alpha-specific mRNA. These findings suggest that ProT alpha may be important in the regulation of the immune response by enhancing MHC class II Ag expression in APC.

Monocyte disorders associated with T cell defects in patients with solid tumors.

T cells proliferate in response to autologous monocytes in the autologous mixed lymphocyte reaction (AMLR). AMLR was found to be impaired in patients with advanced cancer (stages III and IV), whereas normal values were found in the early stages of the disease (stages I and II). Peripheral T lymphocytes from patients with advanced stages also exhibited a decreased ability to produce Interleukin-2 (IL-2) during an AMLR response, whereas production of IL-2 by T cells in stages I and II was comparable to that of normal donors. The impaired IL-2 production by T lymphocytes in the AMLR was associated with high concentrations of soluble interleukin-2 receptor (sIL-2R) in culture supernatants and reduced expression of membrane-bound interleukin-2 receptors (IL-2R) on the same AMLR-activated T lymphocytes. These abnormalities in T cells from cancer patients were demonstrated to be associated with dysfunctions of autologous monocytes. Thus monocytes from patients with advanced cancer exhibited diminished expression of HLA-DR antigens and produced low levels of Interleukin-1 beta (IL-1 beta) and Tumor Necrosis Factor a (TNFa). No changes were detected in the expression of HLA-A, -B, -C antigens. The results presented here demonstrate that decreased in vitro T cell responses may be attributed to monocyte dysfunctions in these patients and provide new information for a better understanding of the impaired T cell function in cancer patients.

Decreased HLA-DR antigen expression on monocytes causes impaired suppressor cell activity in multiple sclerosis.

Mitogen-induced Ts cell activity and (DR) expression on monocytes have recently been shown to be reduced in patients with multiple sclerosis (MS). In our study, T cells were cultured with autologous monocytes in the presence of Con A for 6 days (primary cultures). At the end of the culture T cells were isolated and were 1) tested for surface DR expression and 2) treated with mitomycin C and placed in a secondary culture of allogeneic responder cells and Con A, to test their suppressor function. Treatment of monocytes of the primary culture with an anti-HLA-DR antibody abolished the expression of DR Ag on the T cells as well as their ability to function as Ts cells in the secondary culture. Monocytes from patients with active MS expressed low levels of surface DR Ag and induced reduced amounts of DR Ag on the surface of autologous T cells in primary cultures, which in turn expressed deficient suppressor function when placed in secondary cultures. In contrast, monocytes obtained from patients with inactive disease expressed higher levels of DR and induced higher amounts of DR Ag on autologous T cells, which expressed normal suppressor function. We conclude that the deficient expression of DR Ag on MS monocytes causes reduced suppressor function in activated autologous T cells and may be a primary abnormality in the immunopathogenesis of MS.

Prothymosin-alpha enhances HLA-DR antigen expression on monocytes from patients with multiple sclerosis.

Monocytes from patients with multiple sclerosis (MS) express decreased numbers of class II major histocompatibility complex (MHC) antigens in peripheral blood and are poor stimulators in the autologous mixed lymphocyte reaction (autoMLR). We assessed the effect of prothymosin-alpha (ProT alpha) on the expression of MHC class II antigens by monocytes. Immediately after isolation, monocytes were analyzed for MHC class II antigen expression using a radiolabelled monoclonal antibody specific for a monomorphic determinant on HLA-DR antigens. After incubation with ProT alpha we observed significant increases in HLA-DR antigens on MS monocytes (1.5- to 4-fold increase compared to freshly isolated monocytes). Kinetic analysis revealed that enhancement peaked after 2 days of incubation with ProT alpha. The increase in HLA-DR antigen on MS monocytes resulted in the restoration of the deficient autoMLR in MS patients. This is the first demonstration suggesting a link between HLA-DR antigen expression and cellular immune defects in MS. The significance of low autoMLR responses for T suppressor levels in MS patients is discussed.

Enhancement of human T lymphocyte function by prothymosin alpha: increased production of interleukin-2 and expression of interleukin-2 receptors in normal human peripheral blood T lymphocytes.

The in vitro incubation of phytohemagglutinin (PHA)- or alloantigen-stimulated peripheral blood T cells with prothymosin alpha (ProT alpha) resulted in a marked and reproducible increase in the production of interleukin-2 (IL-2). Incubation of T cells with ProT alpha, in the absence of PHA or alloantigen, failed to induce any production of IL-2. ProT alpha by itself did not exert any IL-2 activity. Finally, ProT alpha was shown to increase the expression of IL-2 receptors on phytohemagglutinin- or alloantigen-activated T cells. These data provide the basis for understanding the in vitro immunoenhancing effects of ProT alpha in cellular immune systems.

Decreased expression of HLA-DR antigens on monocytes in patients with multiple sclerosis.

Immunofluorescence, cell binding assays and enzyme immunoassays were used to investigate the expression of class II major histocompatibility antigens on peripheral blood monocytes in 67 patients with multiple sclerosis. Monocytes from patients with active disease expressed fewer HLA-DR molecules on their surface than normal monocytes; furthermore the percentage of cells which exhibited detectable amounts of surface HLA-DR antigens was decreased in patients with active multiple sclerosis. During the inactive stage of the disease both deficiencies were milder, probably representing secondary pathogenetic phenomena. Quantitation of monocyte surface HLA-DR antigen expression could be valuable in assessing the clinical disease activity. The demonstration of a molecular defect in patients with multiple sclerosis will improve our understanding of the pathogenesis of the disease.

Peptides of myelin basic protein stimulate T lymphocytes from patients with multiple sclerosis.

Peripheral blood T lymphocytes from patients with multiple sclerosis (MS) and other neurological diseases (OND) were tested for primary in vitro proliferation in response to four synthetic peptides derived from the sequence of human myelin basic protein (HuMBP) and to HuMBP 45-89 peptide fragment, using a [3H]thymidine incorporation assay. The synthetic peptides used corresponded to residues HuMBP 15-31, 75-96, 83-96 and 131-141 of human myelin basic protein. Significant proliferation of T lymphocytes to peptides was noted only in the MS group (with the exception of peptide 131-141): the majority of control subjects and OND patients did not respond to the above-mentioned peptides. The sensitized T lymphocytes in MS patients displayed the inducer/helper phenotype and required autologous monocytes for optimal proliferation. An anti-HLA-DR monoclonal antibody, directed against a monomorphic determinant of DR molecules, was able to block the responses in a dose-dependent fashion. These results suggest that autoimmune inducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demyelination process in patients with MS. Furthermore, our data demonstrate that monocytes and HLA-DR molecules are essential for activation of these cells. Finally primary in vitro T cell proliferation to HuMBP synthetic peptide may be used as an additional diagnostic test in MS.

Enhancement of human T lymphocyte functions by prothymosin alpha. I. Augmentation of mixed lymphocyte culture reactions and soluble protein-induced proliferative responses.

Prothymosin alpha (ProT alpha), a 115-amino-acid thymic polypeptide, was tested for its effect on soluble antigen, allo- and auto-antigen-induced human T-cell proliferation. ProT alpha enhanced the secondary T-cell proliferative response to ovalbumin (OVA)- and keyhole limpet haemocyanin (KLH)-pulsed antigen-presenting cells (peripheral blood monocytes). Maximum enhancement (20-fold for OVA and 23-fold for KLH) occurred when suboptimal concentrations of either OVA or KLH were employed. Subset depletion experiments showed that the helper/inducer T-cell subpopulation was responsible for the observed enhancement. In the mixed lymphocyte reaction (MLR), ProT alpha enhanced autoantigen- (autoMLR; 9- to 14-fold) as well as the alloantigen- (alloMLR; 8- to 10-fold) induced T-cell proliferation when suboptimal ratios of the participating cells were used. Preincubation of the stimulating (autologous or allogeneic monocytes) with ProT alpha induced significantly higher T-cell proliferation in both primary and secondary MLR responses as compared to that induced by non-treated monocytes. In contrast, T lymphocytes pre-incubated with ProT alpha did not show enhanced proliferative activity when tested subsequently in the MLR. Suboptimal numbers of T cells exhibited high proliferative activity when pre-incubated with ProT alpha in the presence of autologous monocytes. These studies suggest that ProT alpha potentiates T-cell proliferative responses not directly, but via monocytes which are included in the cultures either as antigen-presenting cells or accessory and/or stimulator cells. The importance of ProT alpha in pathologically occurring defective cellular immune response systems discussed.

Mechanism of action of prothymosin alpha in the human autologous mixed lymphocyte response.

Prothymosin alpha(Prot alpha), an immunologically active polypeptide derived initially from rat thymus, and now pig thymus, was tested for its effect on autoantigen-induced human T cell proliferation in vitro. Pig ProT alpha was found to enhance the autologous mixed lymphocyte response (auto-MLR). Optimum enhancement was achieved at doses which varied among different donors. Treatment of the stimulatory monocytes with ProT alpha resulted in considerably higher auto-MLR responses as compared to those with non treated monocytes. ProT alpha was without effect on T lymphocytes. In contrast, T lymphocytes exhibited enhanced proliferative activity when treated with ProT alpha in the environment of autologous monocytes. Moreover, supernatants from cultures of monocytes incubated with ProT alpha (ProT alpha-sup) were also shown to enhance the human auto-MLR either after addition in cultures or after preincubation with responder T lymphocytes. In addition, ProT alpha-sup did not demonstrate any detectable interleukin 1 (IL 1) or interleukin 2 (IL 2) - like activity. Furthermore, ProT alpha-sup induced an increase in IL 2 production in auto-MLR cultures. The enhancement of T-cell proliferation and IL 2 production by ProT alpha-sup was maximal when this material was added at the beginning of the auto-MLR, and no effect of ProT alpha-sup was seen if the latter was added 3 days after initiation of the culture. Finally, Prot alpha-sup was also shown to increase the expression of IL 2 receptors on T lymphocytes activated in the auto-MLR. These studies suggest that ProT alpha enhances the human auto-MLR through ProT alpha-sup which is released after interaction of monocytes with ProT alpha ProT alpha-sup then increases directly T lymphocyte proliferation by elevating IL 2 production and expression of IL 2 specific receptors on autoactivated T lymphocytes.

Monocyte defect causes decreased autoMLR in multiple sclerosis patients.

We have recently demonstrated that peripheral blood monocytes from patients with multiple sclerosis (MS) have a defect in stimulating autologous and allogeneic T lymphocytes. This defect was found to correlate with disease activity. We report here that T4+ cells from MS patients proliferate weakly in the autologous mixed lymphocyte reaction (autoMLR). Furthermore, we provide direct proof that the depressed T4+ cell proliferation is due to the monocyte functional (stimulatory) defect.

Multiple sclerosis: II. Effects of prothymosin alpha on the autologous and allogeneic MLR in patients with multiple sclerosis.

We have recently demonstrated that peripheral blood monocytes from patients with multiple sclerosis (MS) have a defect in stimulating autologous and allogeneic T lymphocytes. This defect was found to correlate with disease activity. In this report we demonstrate that prothymosin alpha (ProT alpha), a rat thymus fraction 5 polypeptide, restores the MS monocyte stimulatory defect. The concentrations of ProT alpha which induced optimal enhancement of the mixed lymphocyte responses (MLR) were significantly higher when monocytes from patients with active disease were used as stimulators than when monocytes from patients with inactive disease were used. T4+ cells tested with autologous stimulatory monocytes harvested from an inactive stage of MS exhibited considerably higher proliferative responses than when stimulated with autologous monocytes obtained from an acute relapse. The decreased autologous proliferation of T4+ cells in MS patients was restored to normal levels after preincubation with ProT alpha in the environment of autologous monocytes. Our results demonstrate that ProT alpha is capable of fully restoring the deficient stimulatory function of MS monocytes and monocyte-associated functional defects of MS-derived T4+ cells.

Immunoregulatory effects of fraction 5 thymus peptides. I. Thymosin alpha 1 enhances while thymosin beta 4 suppresses the human autologous and allogeneic mixed lymphocyte reaction.

Thymosin alpha 1 and thymosin beta 4, two peptides isolated from preparations of calf thymus fraction 5, were tested in the human mixed lymphocyte reaction (MLR). Thymosin alpha 1 was found capable of enhancing both the allogeneic and autologous MLR. On the contrary, thymosin beta 4 suppressed MLR proliferative responses. Study of the responses of the T cell subpopulations revealed that T4+ (helper/inducer) cells but not T8+ (suppressor/cytotoxic) are responsible for the enhanced, proliferative response to allo- and autoantigens in the presence of thymosin alpha 1. Both the autologous and the allogeneic proliferative responses of either T4+ cells or T8+ cells were not influenced by the addition of thymosin beta 4 in the cultures. However, when T4+ and T8+ subsets were cocultured, thymosin beta 4 was capable of activating T8+ cells to suppress the allogeneic and the autologous proliferative response of T4+ cells. These studies show that thymosin fraction 5 peptides exert immunoregulatory effects on the human MLR proliferative responses in vitro.

Multiple sclerosis: I. Monocyte stimulatory defect in mixed lymphocyte reaction associated with clinical disease activity.

To investigate whether abnormalities of cellular immune responses are associated with multiple sclerosis (MS), we tested peripheral blood mononuclear cells (PBMC) or T cells, and monocytes from MS patients as responder and stimulatory cells respectively, in the allogeneic (allo-MLR) and autologous mixed lymphocyte reaction (auto-MLR). We found that PBMC or T cells from all MS patients were able to develop strong proliferation against allogeneic monocytes derived from normal individuals. Moreover, the capacity of monocytes from MS patients to act as accessory cells for autologous T cells in the allo-MLR was indistinguishable from that of normal donors. In contrast, monocytes from patients with active MS were not able to stimulate responder cell proliferation either in allo-MLR or in auto-MLR. This monocyte defect was partially restored in the inactive stage of the disease. In conclusion our results show that the stimulatory capacity of monocytes from MS patients in the MLR is closely associated with the clinical stage of MS. The observed monocyte defect may be helpful in understanding the pathogenesis of MS and can be used in evaluating the outcome of the disease activity.

Prothymosin alpha restores the depressed autologous and allogeneic mixed lymphocyte responses in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by a variety of profound T-cell abnormalities among which are decreased autologous and allogeneic mixed lymphocyte reactions (auto-MLR and allo-MLR, respectively). In a group of 10 patients with SLE, the mean auto-MLR and allo-MLR responses, tested by tritiated thymidine incorporation, were significantly decreased. If optimal doses of highly purified prothymosin alpha (ProT alpha) were present during the auto- of allo-MLR, the T-cell proliferative responses of SLE patients were increased to normal levels. ProT alpha had more pronounced enhancing effect in patients than in normal individuals. Among patients, ProT alpha was more effective in those who had active disease and low proliferative responses. These results demonstrate that ProT alpha can fully restore the deficient T-cell proliferative responses in auto- and allo-MLR in patients with SLE. ProT alpha, or a certain peptidic fragment of it, could prove potentially useful in the treatment of SLE.