Familial CD8 deficiency due to a mutation in the CD8 alpha gene.

CD8 glycoproteins play an important role in both the maturation and function of MHC class I-restricted T lymphocytes. A 25-year-old man, from a consanguineous family, with recurrent bacterial infections and total absence of CD8(+) cells, was studied. Ab deficiencies and ZAP-70 and TAP defects were ruled out. A missense mutation (gly90-->ser) in both alleles of the immunoglobulin domain of the CD8 alpha gene was shown to correlate with the absence of CD8 expression found in the patient and two sisters. Conversely, high percentages of CD4(-)CD8(-)TCR alpha beta(+) T cells were found in the three siblings. A novel autosomal recessive immunologic defect characterized by absence of CD8(+) cells is described. These findings may help to further understanding of the role of CD8 molecules in human immune response.

Node-negative breast cancers with p53(-)/HER2-neu(-) status may identify women with very good prognosis.

BACKGROUND: The contribution of p53 and HER-2/neu to the management of node-negative breast cancer (NNBC) could be improved by combining their results. MATERIAL AND METHODS: We studied paraffin-embedded primary tumors for p53 (BP-53-12-1) (n=57) and HER2/neu (pAB1) (n=63) from NNBC patients. The results were grouped in a negative (p53(-)/neu(-)) versus a positive group (one or both overexpressed). The association between both groups (negative and positive) and clinicopathologic parameters, S-phase fraction and DNA ploidy, and patients' outcome, was analyzed. RESULTS: In 28% of the tumors p53 was overexpressed, and HER2/neu in 11%. Sixty-five percent (37 out of 57) were p53(-)/neu(-), and 35% overexpressed one (31.5%) or both (3.5%) oncoproteins. Significant correlations were found between p53(-)/neu(-) tumors and age greater than 50 (p=0.003), S-phase fraction lower than 7 (p=0.03), and positive estrogen receptor contents (p=0.049). Actuarial 5-year disease-free and overall survival for p53(-)/neu(-) tumors were 88% and 97%, respectively, versus 50% and 66%, for tumors overexpressing one or both oncoproteins (p=0.004).

CCR5 genotype and HIV-1 infection in perinatally-exposed infants.

The CCR5 chemokine receptor is required by non-syncytium HIV-1 strains to infect target cells. A 32 base pair deletion (delta32) in the CCR5 gene causes a structural CCR5 modification that does not permit HIV-1 entry into cells. The rate of the CCR5 delta32 was investigated in 137 children born from HIV-infected mothers. Overall, five (10.6%) of 47 HIV-infected infants showed the defect in heterozygosis vs. eight (8.9%) of 90 uninfected children. No CCR5 delta32 homozygotes were found. Interestingly, among infected children, five (21.7%) of 23 showing a slow disease progression were heterozygous for the CCR5 delta32, meanwhile none of the 24 infants with rapid disease course had the deletion (P = 0.022). In conclusion, the CCR5 delta32 defect does not protect against vertical HIV-1 transmission, but is associated with a delayed disease progression in HIV-infected children.

Stability of amphotericin B in an extemporaneously prepared i.v. fat emulsion.

The stability of amphotericin B in an extemporaneously prepared i.v. fat emulsion was studied. Admixtures of amphotericin B 0.5, 1, and 2 mg/mL were prepared by adding 10, 20, and 40 mL of amphotericin B 5 mg/mL to 90, 80, and 60 mL, respectively, of 20% fat emulsion. The admixtures were stored in glass vacuum containers at 20-25 degrees C and exposed to fluorescent light, 20-25 degrees C and protected from light, or 4-8 degrees C and protected from light. A sample was withdrawn from each container at 0, 4, 12, and 24 hours and at 2, 4, 7, and 15 days for analysis of amphotericin B concentration by high-performance liquid chromatography and for visual evaluations; these samples were immediately frozen until analyzed. A sample was withdrawn from one container of amphotericin B 1 and 2 mg/mL for each storage condition at 0, 7, and 15 days for immediate determination of particle-size distribution with a fluorescinated-antibody cell sorter. Amphotericin B 0.5 mg/mL in 20% fat emulsion was stable for one week under all the storage conditions. Amphotericin B in the 1- and 2-mg/mL admixtures was stable for up to four days at 20-25 degrees C exposed to fluorescent light, and for up to one week at 20-25 degrees C protected from light and at 4-8 degrees C protected from light. There was no visible evidence of incompatibility. There were no substantial changes in particle-size distribution for the 1-mg/mL admixtures; appreciable changes were detected for the 2-mg/mL admixtures. Amphotericin B 1 and 2 mg/mL was stable in 20% fat emulsion for four days at 20-25 degrees C exposed to fluorescent light and for seven days at 20-25 degrees C protected from light or at 4-8 degrees C; amphotericin B 0.5 mg/mL was stable in 20% fat emulsion for seven days under the three storage conditions.

[Immunologic prognostic parameters in infection by the human immunodeficiency virus]. / Parámetros inmunológicos pronósticos en la infección por el virus de la inmunodeficiencia humana.

BACKGROUND: To evaluate the use of quantitative and functional immunologic parameters as prognostic markers of infection by the human immunodeficiency virus (HIV). METHODS: The number of CD4 and CD8 lymphocytes, the CD4/CD8 ratio, the percentage of interleukin 2 (IL-2) receptors, the response to phytohemaglutinin (PHA) and the production of interferon tau (IFN-tau), were analyzed in 85 patients with HIV infection: 14 with acquired immunodeficiency syndrome (AIDS) (stage IV), 16 with persistent generalized adenopathies (stage III), and 55 asymptomatic patients (stage II). Similarly, a control group of 35 blood donors with negative HIV serology was studied. RESULTS: Over a period of 30 months, 17 patients (5 of stage III and 12 of stage II) evolved to stage IV. In a multivariate analysis the decrease in the number of CD4 lymphocytes in stage II and the decrease in the production of IFN-tau in stage II and III were the parameters associated with progression to stage IV. CONCLUSIONS: The decrease in the production of interferon tau in patients with infection by the human immunodeficiency virus in stage II and III as well as the decrease in the number of CD4 lymphocytes in stage II are prognostic factors associated with the evolution to stage IV of the infection.

Evolution of immunological abnormalities in HIV infection by vertical transmission.

Forty-four children infected through vertical transmission, from a total of 146 born to HIV-positive mothers, were studied. Immunological data were analysed and compared with those of the noninfected children. Two transmission patterns emerge from the clinical and immunological characteristics: (i) infants infected during pregnancy with severe immunodeficiency and clinical manifestations before the age of 1 year, and (ii) children probably infected perinatally, who have better clinical outcome. Immunological data are important for prognosis and early therapeutic protocols to be established.

Chronic granulomatous disease in pediatric patients: 25 years of experience.

INTRODUCTION: Chronic granulomatous disease (CGD) is an uncommon primary immune deficiency (affecting 1/200,000 newborn infants) caused by a defect in phagocyte production of oxygen metabolites, and resulting in bacterial infections produced by catalase-positive microorganisms and fungal diseases that occasionally may prove fatal. METHODS: A review is made of the clinical records of 13 pediatric patients diagnosed with CGD between 1980 and 2005. RESULTS: All patients were males. The mean age at diagnosis was 36 months. The clinical manifestations at the time of diagnosis comprised the following: Abscesses or abscessified adenopathies 4/13 (Staphylococcus aureus (2), Serratia liquefaciens, S. marcescens and Klebsiella sp.), pneumonia 3/13 (Rhodococcus equi, Salmonella typhimurium plus Pneumocystis jiroveci), osteomyelitis 1/13 (Aspergillus sp.), sepsis 1/13 (S. aureus), urinary infection 1/13 (Klebsiella sp.), severe gastroenteritis 1/13, oral aphthae 1/13 and Crohn-like inflammatory bowel disease 1/13. The diagnosis was initially established by the nitroblue tetrazolium test, and confirmed by flow cytometry 10/13 and genetic techniques (gp91) 9/13. In the course of these disease processes there were 88 infections: abscesses (n = 26), lymphadenitis (n = 12), pneumoniae (n = 10), gastroenteritis (n = 7), sepsis (n = 6), osteomyelitis (n = 3) and others (n = 24). As to the germs isolated, the frequency distribution was as follows (n = 49): Aspergillus sp. (n = 10), Staphylococcus sp. (n = 7), Salmonella sp. (n = 6), Serratia sp. (n = 5), Pseudomonas aeruginosa (n = 4), Klebsiella sp. (n = 4), Proteus sp. (n = 3), Leishmania sp. (n = 2) and others (n = 8). IFN-gamma was administered in 7/13 cases, and itraconazole in 9/13; all received cotrimoxazole. There were four deaths, with one case each of sepsis due to gramnegative bacterial infection; disseminated aspergillosis; visceral leishmaniasis and hemophagocytosis; and post-kidney transplant complications. CONCLUSIONS: Clinical suspicion and flow cytometry are the keys for diagnosis of CGD and detection of carrier relatives. Specific prophylactic measures and medical controls are required to prevent serious infections. IFN-gamma has been used intermittently, though its effectiveness is controversial.

Immunological abnormalities in pediatric AIDS.

HIV infection of a fetus from an infected mother has severe immunological implications because it destroys still immature immune system. The abnormalities observed are precocious in the infants. When the infection occurs in perinatal period, clinical and immunological manifestations can present themselves at the age of 2-5 years. Two patterns of HIV infection can be distinguished. Humoral immunodeficiency is present in a high proportion of patients and leads to repeated bacterial infections and progression of the disease. Immunoglobulin substitution therapy improves clinical manifestations and can help to avoid viral replication.

Development of a common variable immunodeficiency in IgA-deficient patients.

IgA deficiency (IgA-D) and common variable immunodeficiency (CVID) are two primary immunodeficiencies that share clinical features. Occasionally, both diseases have been diagnosed in the same family, which suggests the existence of some common pathogenic mechanism, but progression from IgA-D to CVID has rarely been documented. We report three cases of CVID diagnosed 1 to 12 years after IgA-D was detected. Two of these patients presented autoimmune diseases followed by a progressive decline in IgG levels. They are currently on intravenous immunoglobulin therapy with complete remission of their autoimmune and infectious symptoms.

Very late presentation of vertically transmitted HIV-1 infection.

We describe a 13-year-old boy with a very late presentation of vertically transmitted HIV-1 infection. The mother, an intravenous drug user before pregnancy, was diagnosed with AIDS in 1987 when the boy was 6 years old. HIV infection in her son was never suspected or investigated. No other risk factors for this infection can be attributed to the boy. On diagnosis of the infection the boy had moderately severe respiratory symptoms, as classified in category B2 of the 1994 paediatric HIV infection definition, and virological replicative kinetics and the phenotype have been determined. Standard AZT therapy has improved the clinical symptoms, with negativization of plasma p24 Ag and HIV RNA. Clinicians should be aware of this form of presentation of HIV-1 infection to avoid further delay of proper therapy.

Effects of anti-sense oligonucleotides directed toward dihydrofolate reductase RNA in mammalian cultured cells.

The effect of incubations with anti-sense phosphorothioate oligonucleotides directed toward sequences of dihydrofolate reductase (DHFR) RNA has been tested on Chinese hamster ovary cells. The selected targets were the 5'-untranslated region, the translational start, the splice sites and branch point of intron I and polyadenylation regions 1 and 3 of the DHFR RNA. To introduce the oligonucleotides, the cationic liposome DOTAP was used. The oligonucleotides most effective at causing cytotoxicity were ATNL and DTNL, both directed toward the translation-start site, at a range of concentrations between 1 and 4 microM. The minimum time for the oligonucleotide to exert its full cytotoxic effect was 3 days. Excess of oligonucleotide diminished the cytotoxic effect. Oligonucleotide uptake was monitored by the incorporation of [32P]- or fluorescein-labeled oligonucleotide and was found to depend on liposome and oligonucleotide concentrations and duration of incubation. Formation of in vitro complexes between the oligonucleotide and the liposome was also studied. Cytotoxicity was observed when the oligonucleotide was incubated with cell lines containing either the endogenous gene or co-transfected DHFR minigenes. Cell incubation with ATNL caused a time-dependent decrease in the levels of DHFR mRNA and enzymatic activity. Moreover, a cell line bearing amplification at the dhfr locus was equally affected by the action of ATNL. Human hepatoma cells were also affected by treatment with the counterpart of ATNL in the human DHFR mRNA sequence. Our results set the basis for a possible cancer therapy with anti-sense oligonucleotides using DHFR as the target.

DNA ploidy study of resected hepatocellular carcinoma in cirrhotic liver.

BACKGROUND/AIMS: Results of several studies on DNA ploidy as a prognostic indicator in hepatocellular carcinoma are contradictory. The present study analysed the correlations between DNA ploidy of resected hepatocellular carcinoma and tumour characteristics, tumour recurrence, risk factors and survival. METHODS: Tumoural DNA ploidy of hepatocellular carcinomas from 37 patients with cirrhosis who underwent curative tumour resection was studied by flow cytometry. RESULTS: A diploid pattern was found in 23 hepatocellular carcinomas (62.2%) and an aneuploid pattern in 14 (37.8%). The tumour recurrence rate did not differ statistically between diploid (69.6%) and aneuploid (50%) hepatocellular carcinomas. The only prognostic variable with significant difference in DNA pattern was the histologic tumour type; the majority of non-trabecular tumours were aneuploid while most trabecular hepatocellular carcinomas had a diploid DNA pattern. Actuarial survival at 1, 2, 3 and 4 years of patients with diploid and aneuploid tumours was 69.6%, 40.6%, 16.2% and 0%, and 69.3%, 59.4%, 49.5% and 32.9%, respectively (log rank p = 0.1927). CONCLUSION: These results indicate that DNA ploidy has no prognostic value in hepatocellular carcinoma.

Cell-growth regulation of the hamster dihydrofolate reductase gene promoter by transcription factor Sp1.

The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains cis-acting elements for the transcription factors Sp1 and E2F. Given the ability of Sp1 to activate the dhfr promoter, we have evaluated the contribution of Sp1 to the cell-growth regulation of the dhfr gene. Using gel-mobility assays performed with DNA probes from the minimal promoter of the hamster dhfr gene and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Sp1 to the dhfr promoter is cell-growth-phase regulated. Accordingly, dhfr transcription and mRNA levels in K1 cells increase upon serum stimulation. Cytological detection of Sp1 by immunofluorescence reveals a decrease of this protein in the process leading to the G0 state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by western blot analysis. It is concluded that Sp1 progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Sp1 in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr-negative cells transfected with dhfr plasmids carrying either the wild-type or mutated Sp1-binding or E2F-binding sites in the dhfr minimal promoter.

Enumeration of CD4(+) T-cells in the peripheral blood of HIV-infected patients: an interlaboratory study of the FACSCount system.

The aim of the present study was to assess the interlaboratory reproducibility of the FACSCount system for the enumeration of peripheral blood (PB) CD4(+) T-cells. In each of the seven participating centers, both previously stained and unstained PB samples (n = 49) were received and either analyzed or stained and then analyzed. Interlaboratory reproducibility was checked in two different groups of centers (n = 3 and n = 4) where the study was performed in parallel. In addition, both the intralaboratory precision and accuracy of this system were analyzed in comparison with results obtained with conventional flow cytometry. Accordingly, upon comparing both methods, a high degree of correlation was observed in the total number of CD3(+) T-cells (coefficient of correlation of 0.9750 +/- 0.0184, slope of the best linear fit: 0. 9214 +/- 0.0311, y-intercept of 12 +/- 47) as well as in the number of CD3(+)/CD4(+) (coefficient of correlation of 0.9794 +/- 0.1457, slope of the best linear fit: 0.9463 +/- 0.0753, y-intercept of -11 +/- 36) and CD3(+)/CD8(+) (coefficient of correlation of 0.9728 +/- 0.0192, slope of the best linear fit: 0.9682 +/- 0.0735, y-intercept of 7 +/- 95) major subsets. In addition, low coefficients of variation (CV) were obtained for replicates, indicating the method's high degree of accuracy. The present study shows that with respect to the interlaboratory reproducibility reported for most techniques used for the enumeration of PB CD4(+) T-cells, the FACSCount system results in data with much lower coefficients of variance (CVs) (mean CV of less than 10%). Upon measuring the impact on results of different variables associated with either sample preparation or data acquisition and analysis, our study clearly shows that data acquisition and analysis does not influence the results by increasing variability since the coefficients of variation obtained for samples prepared in the same laboratory under the same conditions and read in different laboratories with different instruments were identical to those obtained for the replicates of the same samples read in each individual center. In contrast, interlaboratory variability, although low, significantly increased when sample preparation was carried out in different laboratories, suggesting that pipetting still represents the major source of variability in the FACSCount system.

Kinase mutant Btk results in atypical X-linked agammaglobulinaemia phenotype.

X-linked agammaglobulinaemia (XLA) is a B cell humoral abnormality arising from mutations in the gene encoding Bruton's tyrosine kinase (Btk). The phenotype of XLA can be variable, with some individuals having a less severe immunophenotype, although in most cases this cannot be correlated with the Btk mutation or expression of Btk protein. In this study we describe clinical and immunological heterogeneity within the same pedigree. Analysis of the genetic defect identified a missense mutation in the kinase domain of Btk which, unusually, preserved Btk protein expression but at reduced levels, and also considerably diminished autophosphorylation activity. Structural analysis of the effect of this mutation on the kinase domain suggests that this mutation is not an integral part of the ATP or substrate binding domains but may affect the interaction of the kinase domain with its own kinase domain and other substrates. Together, these data may provide an explanation for the variable XLA phenotype.

Evaluation of the putative role of C-C chemokines as protective factors of HIV-1 infection in seronegative hemophiliacs exposed to contaminated hemoderivatives.

BACKGROUND: Overproduction of beta-chemokines and genetic variations in chemokine receptors have been correlated with protection against infection by HIV-1 or slow progression to AIDS in infected individuals. STUDY DESIGN AND METHODS: The protective role of chemokines and their receptors was evaluated in a group of seven uninfected (seronegative) hemophiliacs transfused with hemoderivatives presumably contaminated with HIV-1. This group was compared to a group of seven infected (seropositive) hemophiliacs and a group of healthy donors (controls). The CD4+ cell count, intracellular cytokine levels, beta-chemokine levels in plasma, beta-chemokine production by PBMNCs, and expression of chemokine receptors CCR5 and CXCR4 in CD4+ cells were evaluated. The occurrence of protective genotypes in CCR5, CCR2b, and SDF-1 (stromal cell-derived factor 1) genes and susceptibility to infection by HIV-1 were also studied. RESULTS: Significant differences in the production and plasma levels of beta-chemokines among the three groups were not detected. Lower IL-2 and IFN-gamma production was observed in the uninfected exposed hemophiliacs than in the controls. Genetic analysis of CCR5, CCR2b, and SDF-1 showed several polymorphisms associated with resistance in some HIV-exposed uninfected hemophiliacs. However, these genetic features cannot explain the protection of all exposed hemophiliacs. In fact, only one patient, carrying two copies of CCR5 from which 32 bp was deleted, showed low CCR5 expression and low susceptibility to infection by a CCR5-using HIV-1 strain. In contrast, PBMNCs from all other individuals supported infection in vitro by both CCR5- and CXCR4-using HIV-1 strains. CONCLUSION: It is not possible to assign to beta-chemo-kines and polymorphisms in chemokine receptors a central role in preventing HIV-1 infection. Natural protection against HIV-1 infection is likely to be due to a multiplicity of factors.

Vertical HIV-1 transmission correlates with a high maternal viral load at delivery.

Transmission of HIV-1 from an infected mother to her child occurs in around 20% of cases. Although maternal, immunological, and virological factors have been implicated in transmission, clear association is not yet well defined. For this reason, we have conducted a study to determine the relative contribution of the above-mentioned factors with special emphasis on quantitative viral load. We studied 67 HIV-1-infected mothers during pregnancy and labor and their 69 newborns (two sets of twins) from two university hospitals in Barcelona. Plasma and cell samples were collected at delivery between January 1992 and May 1994, and HIV-1 RNA and p24 in plasma, CD4 cell counts, and tissue culture infectious doses (TCID) were measured. Diagnosis of infection in children was based on persistence of anti-HIV-1 antibodies at 18 months of age, a positive HIV-1 culture or polymerase chain reaction in two separate samples, or presence of signs or symptoms of AIDS before 18 months of age. Results showed a very high relationship between > 10(5)/ml viral RNA copies (odds ratio [OR] 22, 95% confidence interval [CI] 4.4-119.2, p < 0.00001), > 0.5 TCID (OR 17, 95% CI 2.1-139.7, p = 0.001), CDC B + C (OR 3.5, 95% CI 0.98-12.5, p = 0.055), < 400 CD4 cells (OR 4.1; 95% CL 1.1-15.4, p = 0.01) and transmission of HIV-1. In this study, a strong association between mother-to-child transmission of HIV-1 and a high maternal viral RNA load in plasma at delivery is demonstrated. Viral load, which is related to clinical and immunological status in the mother, is the main contributing factor for HIV-1 vertical transmission, and these findings may have global and even individual therapeutic implications.