Mutation of the matrix metalloproteinase 2 gene (MMP2) causes a multicentric osteolysis and arthritis syndrome.

The inherited osteolyses or 'vanishing bone' syndromes are a group of rare disorders of unknown etiology characterized by destruction and resorption of affected bones. The multicentric osteolyses are notable for interphalangeal joint erosions that mimic severe juvenile rheumatoid arthritis (OMIMs 166300, 259600, 259610 and 277950). We recently described an autosomal recessive form of multicentric osteolysis with carpal and tarsal resorption, crippling arthritic changes, marked osteoporosis, palmar and plantar subcutaneous nodules and distinctive facies in a number of consanguineous Saudi Arabian families. We localized the disease gene to 16q12-21 by using members of these families for a genome-wide search for homozygous-by-descent microsatellite markers. Haplotype analysis narrowed the critical region to a 1.2-cM region that spans the gene encoding MMP-2 (gelatinase A, collagenase type IV; (ref. 3). We detected no MMP2 enzymatic activity in the serum or fibroblasts of affected family members. We identified two family-specific homoallelic MMP2 mutations: R101H and Y244X. The nonsense mutation effects a deletion of the substrate-binding and catalytic sites and the fibronectin type II-like and hemopexin/TIMP2 binding domains. Based on molecular modeling, the missense mutation disrupts hydrogen bond formation within the highly conserved prodomain adjacent to the catalytic zinc ion.

Progesterone does not potentiate the acrosome reaction in human spermatozoa: flow cytometric analysis using CD46 antibody.

The study was designed in order to investigate the action of progesterone on the spontaneous and ionophore-induced human spermatozoa acrosome reaction in vitro. The principle of the assay system is flow cytometric analysis of CD46 antibody binding to the inner acrosomal membrane. The technique is a simple and objective method of analysis, allowing fluorescent analysis of a large segment (5000 spermatozoa) of the spermatozoa population under investigation, with concomitant isolation of the live fraction of the spermatozoa population. Four concentrations of progesterone (1, 25, 50, and 100 microg/ml) were examined for their effects on spermatozoa capacitated for 4 and 24 h. In addition, motility parameters were examined by the CellSoft 2000 automated semen analyser system. Analysis of variance revealed that progesterone had no effect on either the spontaneous acrosome reaction or the ionophore-induced acrosome reaction at both 4 h and 24 h of spermatozoa capacitation times. Further, no effects on sperm motility parameters or on spermatozoa viability could be attributed to progesterone. We therefore conclude that progesterone has no objectively measurable effects on either the sperm acrosome reaction or sperm motility parameters, as measured in normal sperm populations.

Nigella sativa: effect on human lymphocytes and polymorphonuclear leukocyte phagocytic activity.

The effects of Nigella sativa (N. sativa) seeds and their soluble fractions were studied in vitro on lymphocyte response to different mitogens and on polymorphonuclear leukocyte phagocytic activity. No stimulatory effect of N. sativa was detected on lymphocyte response to phytohemagglutinin, concanavalin-A or pokeweed mitogen. A stimulatory effect of N. sativa was noticed on the lymphocyte response to pooled allogeneic cells. This effect was more pronounced when the low molecular weight (< 10 kDa) fraction was used and varied from one normal individual to another (25% to 825%). N. sativa enhanced the production of interleukin-3 by human lymphocytes when cultured with pooled allogeneic cells or without any added stimulator. N. sativa did not, however, enhance or suppress interleukin-2 secretion by mitogen activated peripheral blood mononuclear cells. Interestingly, N. sativa increased interleukin-1 beta, suggesting therefore, that it has an effect on macrophages. It also suppressed the leukocyte chemiluminescence activity using phorbol myristate acetate and Zymosan as stimulants. No effect of N. sativa or its fractions was, however, noticed on bacterial phagocytosis or killing when Staphylococcus aureus was used, indicating that the decrease in chemiluminescence activity in the presence of N. sativa is not relevant to the bactericidal activity.

Evaluation of anti-ds DNA antibody measurement by using commercial kits for use in a clinical laboratory.

Three hundred and seventy-six consecutive antinuclear antibody-positive sera were tested for anti-ds DNA antibody by using three commercial kits which use 125 I recombinant DNA (radioimmunoassay), highly purified calf thymus DNA (enzyme linked immunosorbent assay) and Crithidia lucilliae (immunofluorescence assay) as substrates. All patients' sera, after reviewing medical records, were classified into three broad groups: Group I (systemic lupus erythematosus), Group II (rheumatic diseases and rheumatoid arthritis), and Group III (nonspecific ANA antibody test positive). A sensitivity, specificity, positive predictive test value and negative predictive test value for Group I against Group II-III (generally these two groups of sera should not show any anti-ds DNA antibody) combined showed for Crithidia lucilliae (IF assay) 58.8%, 93.6%, 82% and 82%, for 125 I recombinant DNA (RIA) assay, 75.8%, 94%, 86.2% and 88.7% and calf thymus highly purified DNA (ELISA) assay using positive cut-off value >100 U/mL, 97.5%, 35%, 42.9% and 24%. The 125 I recombinant DNA (RIA) assay based on the principle of the Farr technique, which is still considered to be the gold standard for anti-ds DNA antibody detection, showed the best specificity and sensitivity among all three methods tested in this study.

Results of 2426 in vitro fertilization cycles from King Faisal Specialist Hospital and Research Centre, 1986-1992.

The report consists of summary in vitro fertilization (IVF) results for the period of 1986 to 1992, concerning 2426 IVF cycles on 954 patients. Tubal factor accounted for 48.7% of cases; male factor, 15.2%; unexplained factors, 15.8%; tubal plus male factor, 12.2%; with 7.7% miscellaneous pathologies. Stimulation regimes were of seven varieties over the period described. All cycles were monitored by transvaginal ultrasound and serum estradiol/LH. All oocyte retrievals were carried out at 34 hours post-HCG. Standard laboratory procedures were utilized for oocyte and pre-embryo culture and a maximum of four embryos were replaced approximately 48 hours after ovum pickup (OPU). Luteal support was by either progesterone suppository or intramuscular injection for 14 days following embryo transfer. Average number of oocytes per retrieval was 7.8, with a fertilization rate of 54% over all groups. Two hundred and sixty-eight pregnancies were initiated (16.1% per embryo transfer [ET]) with 53 of these being biochemical. Pregnancy rates followed a learning curve in the early years with a plateau at 17.8% per ET. Thus, the IVF program has evolved to yield acceptable results after refinement of stimulation regimes, laboratory techniques (especially with regard to sperm preparation), ET techniques and luteal support, thereby tailoring its protocols to suit the local population.

Human leukocyte antigen typing and associated abnormalities in premature ovarian failure.

A case-controlled study was designed to assess the distribution of human leukocyte antigens (HLA) A, B, Cw, DR and DQ, and associated abnormalities in patients with premature ovarian failure (POF). A total of 37 patients in a tertiary care centre were diagnosed as having POF; all were < 37 years old. A subset was HLA-typed. The occurrence of associated diseases in patients with POF was recorded. A comparison of HLA typing was made between patients with POF and 100 organ donors from the same population. No statistically significant difference in the distribution of A, B, Cw, DR and DQ antigens was found between the study and control groups when corrected for the number of antigens tested. The commonest associated abnormality was positive autoimmune screen (43.3%). Abnormal thyroid function tests were detected in 23.3% of the patients. The observed high frequency of associated autoimmune phenomena in patients with POF points to an underlying autoimmune disorder in POF and warrants further studies to unravel the pathology of this condition.

Pentoxifylline potentiates ionophore (A23187) mediated acrosome reaction in human sperm: flow cytometric analysis using CD46 antibody.

The study was designed to investigate the effects of pentoxifylline on the acrosome reaction of human spermatozoa in vitro, and to determine whether the reaction is differently modulated after sperm selection by multiple tube swim-up and Percoll buoyant density centrifugation. The acrosome reaction was induced in vitro by using calcium ionophore (A23187) and was detected by measuring the fluorescence of FITC-conjugated goat anti-mouse immunoglobulin bound to CD46 antibody (which binds to the CD46 receptor site on the inner acrosomal membrane) by flow cytometry. Spermatozoa separated on Percoll displayed significantly lower spontaneous acrosome reactions (P = 0.002) than did those separated by the swim-up technique. Pentoxifylline did not, by itself, induce acrosome reaction, but after induction with ionophore, it significantly increased the reaction (P = 0.003) and this increase was seen to be greater when Percoll separation was used as compared to the swim-up technique (P = 0.0002). We therefore conclude that Percoll selection of motile spermatozoa together with pentoxifylline treatment may be of value in assisted reproductive techniques, as an increased ARIC score arose after both treatments, and that flow cytometry allows a precise and rapid quantification of the acrosome reaction.

Modified whole blood microtechnique for measurement of mitogens and allogeneic lymphocyte stimulated proliferation of human lymphocytes.

A modified micro whole blood technique for the measurement of human lymphocyte proliferative response upon stimulation with commonly used mitogens such as phytohemagglutinin (PHA), concanavalin-A (con-A), pokeweed mitogen (PKW) and pooled allogeneic lymphocytes is described. The modified microtechnique results are comparable to the conventional techniques using density gradient separated lympocyte. The advantage of this micro whole blood technique is the capability of measuring response to common additional equipment is needed. It can easily be adopted by laboratories performing lymphocyte proliferative assay.

Percoll semen preparation enhances human oocyte fertilization in male-factor infertility as shown by a randomized cross-over study.

The aim of this study was to compare two methods of semen preparation: multiple tube swim-up and Percoll separation, using a randomized cross-over clinical study, in which sperm parameters, oocyte fertilization rates, embryo quality and cell stage were analysed. Overall, there was no difference between the two preparation methods in the normozoospermic cycles. In the male-factor cycles, Percoll extracted a higher total number of spermatozoa (P = 0.02), increased the concentration of motile spermatozoa (P = 0.02), increased the total number of motile spermatozoa per sample (P = 0.02), and enhanced the recovery rate of motile spermatozoa (P = 0.04) compared to swim-up. There was a significant improvement in fertilization rates (P = 0.0006), in the percentage of embryos over 2-cell stage on day of transfer (P = 0.004), and in the number of replaced embryos per transfer (P = 0.01) in the Percoll as compared to swim-up cycles. There was no significant difference in embryo quality. We conclude, therefore, that in advanced reproductive procedures where sperm dysfunction exists, semen preparation with Percoll should replace the swim-up technique.

Effectiveness of four intravenous IgG preparations in prevention of phagocytosis of anti-Rh-(D)-coated erythrocytes by mononuclear phagocytes.

Four commercial intravenous immunoglobulin G (IGIV) preparations, Sandoglobulin (Sandoz), Intraglobin (Biotest), Gammonativ (Kabivitrum) and Gammaogard (Kabivitrum) showed percent inhibition of Fc receptor-mediated phagocytosis of anti-Rh(D)-coated erythrocytes as follows: 45 +/- 14 SD, 41 +/- 23 SD, 35 +/- 14 SD, 25 +/- 13 SD, respectively. Pre-incubation of Rh(D)-positive and antibody-coated Rh(D)-positive erythrocytes with IGIV did not lead to any inhibitory effects on phagocytosis. However, preincubation of mononuclear phagocytes with IGIV gave marked inhibition of phagocytosis of antibody-coated erythrocytes. In summary, all four IGIV preparations showed Fc receptor-mediated inhibition of phagocytosis of anti-Rh(D)-coated erythrocytes by mononuclear phagocytes.

Analysis of peripheral blood lymphocyte subsets in normal Saudi males by flow cytometry.

Lymphocyte immunophenotyping using flow cytometer has become an important tool for clinical patient management as well as for research and epidemiological studies. We examined the distribution of CD3 (all T cells), CD4 (T helper/inducer cells), CD8 (T suppressor/totoxic cells), CD16 (natural killer cells) and CD19 (B cells) in 150 healthy Saudi male blood donors using flows cytometry. The two-color labeled cells were analyzed by using the flow cytometer (FACScan, Becton-Dickinson, San Jose, California, USA) and the dual fluorescent subsets were discriminated by Simultest software. The distribution of T lymphocytes, B lymphocytes, and natural killer (NK) cells were similar to those reported in other populations as well as in normal Caucasian expatriate donors (all males) (n = 40) who were included in this study as controls. However, a significantly decreased CD4/CD8 ratio was observed in most Saudi blood donors. These lower ratios were due to decreased CD4 together with an increase in CD8 cells. Significant (P<0.00001) difference in CD4/CD8 ratio in our study may be due to environmental factors such as ultraviolet radiation and stress (heat) as well as some genetic factors.

Anti-tumor cytotoxic potential and effect on human bone marrow GM-CFU of human LAK cells generated in response to various cytokines.

The effects of various recombinant cytokines i.e. IL-1 alpha, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha and GM-CSF used either alone or in combination with IL-2, were investigated in this study. First, their capacity to induce killer cells from human PBL was examined by evaluating the degree of killing of human NK-sensitive K562 or NK-resistant Daudi cells. Second the effects of these cytokines, LAK cells (at 1/1, 2/1, 4/1 ratio LAK effectors/bone marrow cell targets) and of the supernatants from washed killer cell cultures, were examined on the colony forming ability of human bone marrow for GM-CFU in vitro. Various degrees of NK activity against K562 was observed in PBL stimulated with the cytokines, whereas LAK activity was found only with IL-2 alone. Culture of PBL with IL-2 + IL-1 alpha or IL-2 + IL-6 or IL-2 + GM-CSF resulted in the highest LAK killing. However, addition of TNF-alpha, or IFN-gamma to IL-2 in cultures resulted in a significant suppression of LAK cell activity. Addition of IL-1 alpha, IL-2, IL-3, and IL-4 to BM cultures had little or no effect on day 14 GM-CFU, whereas addition of IL-6 and GM-CSF resulted in a stimulatory effect. LAK cells induced with IL-2 alone had no significant suppressive effects on GM-CFU.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphologic immunophenotypic and cytogenetic patterns of adult acute myeloid leukemia in Saudi Arabia.

During a 6-year period we received bone marrow (BM) and peripheral blood (PB) samples from 178 patients with acute myeloid leukemia (AML). All patient BM, and occasionally, PB samples were characterized according to FAB criteria, and by immunophenotyping (IP) and cytogenetics (CG). This report summarizes the findings in the 125 patients who were older than 15 years. Their mean and median ages were 39.4 and 37.0 years. There were 8 (6.4%) M1, 27 (21.6%) M2, 15 (12.0%) M3, 49 (39.2%) M4, 14 (11.2%) M5A, 9 (7.2%) M5B and 2 (1.6%) M6. IP showed that HLA-DR was most strongly and frequently expressed by M1 blasts (53.5%, 86%) and least strongly and frequently expressed by M3 blasts (4.5%, 0%). HLA-DR was also relatively strongly expressed by M4, M5A, M5B (21.5%, 43%; 34.9%, 69%; and 19.2%, 56%, respectively). CD11b was uniformly weakly expressed by all FAB subgroups. CD13 was most strongly and frequently expressed by M4 (20%, 43%), and was relatively weakly and infrequently expressed by the other FAB subtypes (9.5%, 9.2%, 16.4%, 8.4%, 16.3%). CD14 was moderately expressed by M4 (15.2%, 25%) and M5B (14.0%, 22%) and M1 (7.0%, 40%). CD33 was most strongly expressed by M3 blasts (26.3% and 61%), and was most weakly expressed by M5B (10.6% and 22%). Fourteen (11.2%) patients had blasts that showed lymphoid antigens (5 T, 5 B, 5 CALLA) in addition to myeloid characteristics. Fifty-four (51.9%) of 104 patients tested had one or more karyotypic abnormalities, the most frequent of which was 8+. Only the t(15:17) was specific, and was seen in M3. Four patients with anomalous IP had trisomy 21, one of whom also had 11q-. We conclude that Saudi Arabian AML shows FAB patterns similar to patients in the West, and that M3 patients have a characteristic IP and cytogenetic pattern. Apart from this the MIC classification failed to reveal characteristic modes.

Morphological and immunological pattern of blastic transformation in chronic myeloid leukemia in Saudi Arabia: Study of 90 transformations among 248 patients.

We examined the pattern of blastic transformation in 90 of 248 patients (36%) with chronic myeloid leukemia who were seen at the King Faisal Specialist Hospital and Research Centre between 1975 and 1988. The mean and median ages of all patients were 38.2 and 36.0 years, respectively. Four of the 90 transformants (4.4%) presented in blastic transformation, and 86 cases (95.5%) evolved from a well-defined chronic phase. Twenty-nine (32.2%) of the patients underwent lymphoid blastic transformation, while 28 (31.1%) were myeloid, seven (7.8%) were myelomonocytic, four (4.4%) were monocytic or erythroblastic, six (6.7%) were megakaryoblastic, ten (11.1%) were of mixed lineage, and two (2.2%) were unclassifiable. The lymphoid blast cells were uniformly common acute lymphocytic leukemia (i.e., Ia and CD10 positive), whereas the myeloid transformations were predominantly Ia negative. Mixed phenotype blasts were also predominantly Ia positive (i.e., 8 of 10), with varying positively for CD10 and myeloid/monocyctic markers. We conclude that blast crisis in chronic myeloid leukemia occurs in Saudi patients in a pattern similar to that seen in patients elsewhere, and that surface Ia antigen positivity in lymphoblast cells is a reliable marker for differentiating lymphoid from nonlymphoid crisis, in which the Ia antigen is not usually demonstrable.

Prediction of successful embryo implantation by measuring interleukin-1-alpha and immunosuppressive factor(s) in preimplantation embryo culture fluid.

To find a better predictor of pregnancy after in vitro fertilization (IVF), supernatant fluids from embryo culture media were analyzed after 24 hours and 48 hours for the presence of interleukin-1-alpha (IL-1), interleukin-2, and the percent of immunosuppression. The measurements were performed on 108 consecutive IVF cycles between June 1989 and October 1989. The IL-1 level +/- SD in the 24-hour aliquots of the supernatant of embryo culture fluid was 66.2 +/- 10.2 pg/mL in all viable pregnancy cycles and 35.4 +/- 9.01 pg/mL in unsuccessful cycles. The percent of immunosuppression after 24 hours was 22.06% +/- 4.5% in viable pregnancy cycles and 7.3 +/- 5.5% in unsuccessful cycles. The percent of immunosuppression 48 hours after ovum pick-up was generally decreased in all embryo culture fluid, showing 17.5% +/- 4.4% in viable pregnancy cycles and 3.8% +/- 3.6% in unsuccessful cycles. Interleukin-1 levels in the 48-hour aliquots were moderately decreased, being 39.0 +/- 6.3 pg/mL in viable pregnancy cycles and 34.3 +/- 4.7 pg/mL in the unsuccessful cycles. In 24 hours, embryo culture aliquots IL-1 level greater than 60 pg/mL was seen in 17 of 21 (80.9%) pregnancy cycles, and the combined data of IL-1 level greater than 60 pg/mL and/or greater than 20 percent of immunosuppression predicted 21 of 21 (100%) pregnancy cycles.

Immunophenotypic and age patterns of childhood acute lymphoblastic leukemia in Saudi Arabia.

Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood in the West, characteristically showing a peak incidence in children aged between two and five years, and being predominantly of the common ALL (cALL) phenotype. In this article, we examine the hypotheses that ALL is relatively less common among childhood malignancies in Saudi Arabia; that the cALL phenotype is uncommon; that T cell ALL (TALL) is relatively more common. We report that of 163 children with ALL seen at the King Faisal Specialist Hospital and Research Centre, we find that their median age was 5.0 years with a modal value of 3 years, with a range of 4 months to 14 years; that there were 93 cALL patients who were predominantly young (median age 5.0 years). There were 20 (12.3%) patients with TALL, whose median age was 8.5 years, 35 (21.5%) patients who were null cell ALL and whose median age was 6.0 years, 14 (8.6%) patients with B cell ALL whose median age was 9.0 years, and 3 (1.8%) patients with mixed phenotype ALL. We also identify a group of 6 (3.7%) patients whose blasts were CD10 negative and showed B cell differentiation without surface membrane immunoglobulin. We conclude that age and phenotypic characteristics of ALL patients are mainly similar to ALL in the West but that L3 was much more common. A small group of six patients showed unusual B cell phenotype and require further evaluation and analysis.

Mycetoma in Saudi Arabia.

The clinical and microbiological features of 31 cases of mycetoma seen in 8 years at a Riyadh hospital are described. Eighteen were due to Streptomyces somaliensis, 10 to Madurella mycetomatis and one each to Actinomadura madurae, Nocardia asteroides, and an unidentified species of Cladosporium. No immune defects were detected in the patients. Eleven had osteolytic lesions and 17 required surgery. Only seven patients were cured. Streptomyces infections were seen from all parts of Saudi Arabia outside the Rub Al Khali, but Madurella occurred mostly in highland regions where rainfall is higher.

Lineage ambiguity in acute leukemia.

Two cases of acute nonlymphocytic leukemia that showed surface phenotypes characteristic of lymphoid cells are reported. The cases, both involving female patients were studied by a variety of methods including flow cytometry and karyotyping. In Case 1, the patient, a 10-year-old girl, had poorly differentiated myeloblasts (FAB M1), which were weakly positive for Sudan black B (SBB), but negative for alpha naphthyl acetate esterase (NAE) and naphthol ASD chloroacetate esterase (CAE). Myeloperoxidase was demonstrated ultrastructurally in some of the blasts. In Case 2, the 30-year-old patient had typical myelomonocytic leukemia (FAB M4), with SBB-, NAE-, and CAE-positive blasts. Both cases were negative for terminal deoxynucleotidyl transferase. Case 1 was negative for myeloid membrane markers, whereas Case 2 was strongly positive for My7 and My9. Surprisingly, both cases showed significant positivity for B-cell restricted antigens B1, B2, and B4. These findings suggest ambiguous or dual lineage, supporting the concepts that some leukemias could arise from a pluripotent hematopoietic progenitor cell (Case 1) or from cells that though differentiated in some respects, could still preserve some early antigens (Case 2).