Pathological, immunological and molecular diagnosis of rabies in clinically suspected animals of different species using four detection techniques in Jordan.

A total of 29 brain tissue samples (BTS) were examined for rabies infection by different diagnostic techniques. None of the examined brain tissues were presented as a whole intact brain. Twenty-seven brain tissue samples from various animal species - dog (13 cases), cat (one case), fox (one case), pig (one case), cow (three cases), sheep (two cases), goat (one case), camel (one case), horse (one case) and donkey (three cases) - were provided by the Vaccine and Sera Department/Al-Bashir Central Hospital in Amman/Jordan from July 2009 up to May 2010. All these samples were frozen at -20°C, for a period of time and then fixed in 10% formalin after being tested for rabies virus by fluorescence antibody test (FAT). The results showed that 21 (77.77%) of 27 BTS were positive for rabies by FAT. Seventeen samples (58.62%) of 29 were positive by histopathology, 2 (6.90%) were positive by histopathology, immunohistochemistry (IHC) and of those which were fixed for 24h only, and 21 (72.42%) were positive using RT-PCR assay. Five of 29 BTS had no pathological lesions, 17 had Negri bodies and the remaining had non-suppurative encephalitis and necrosis. Thirteen BTS that were diagnosed positive by FAT were also positive by RT-PCR and histopathology, but negative by IHC. Four BTS that were positive by FAT were negative by histopathology, IHC and RT-PCR. Also, 3 BTS (cases 19, 22, and 25) that were negative by FAT were positive by RT-PCR and negative by IHC. One of these was negative, while two were positive by histopathology. Therefore, definitive diagnosis of rabies under these conditions in Jordan needs one or more other diagnostic tests in addition to FAT. Also, freezing and prolonged formalin fixation of BTS is not suitable for the detection of rabies virus antigen using IHC.

Human and animal brucellosis in Jordan between 1996 and 1998: a study.

Between 1996 and 1998, a total of 2,494 samples of blood from humans and animals were collected and tested for brucellosis. This total included 1,594 samples of animal blood, collected from 1,050 sheep from 20 flocks, and 544 goats from eight herds. The serum samples were tested using the Rose Bengal test, the tube agglutination test, the complement fixation test and an enzyme-linked immunosorbent assay. Moreover, a complete history was compiled from each flock/herd. The rate of abortions in sheep due to brucellosis ranged from 0.5% to 56%, with a mean of 33.2%. The goats had a higher abortion rate. Thirty-four aborted sheep foetuses collected from these 20 flocks were bacteriologically and pathologically examined. A pure culture of Brucella melitensis biotype 3 was isolated from 21 of the aborted foetuses. The human blood samples were collected from two groups: first, from 800 apparently healthy people who were reporting to community hospitals for routine health checks and secondly, from 100 people from groups with a high-risk of contracting brucellosis, such as veterinarians, sheep-herders and laboratory technicians. The Brucella antibody titres for the 900 human serum samples were obtained using the microtitre agglutination test. The cumulative percentage of the serum samples showing a titre reading greater than 1:80 was higher in the at-risk group than among the normal population (7% compared to 4.1%). Although these results were not statistically significant, the higher percentage of positive reactors among the high-risk group may indicate an increased risk factor among professional agricultural and veterinary personnel in Jordan. It was concluded that brucellosis is common in sheep and goats in Jordan, subjecting the human population to high risks. Brucella melitensis Rev. 1 vaccination has been internationally recognised as the key to successfully controlling the disease. All animals in Jordan were repeatedly vaccinated between 1996 and 1998 on a trial basis, using a reduced dose of 1 x 10(5) colony-forming units (CFU). Cumulative data on the annual rate of human cases of brucellosis indicate that fewer people are affected each year. The same is true for the rate of abortions in animals. Such evidence strongly suggests that the vaccination programme has been successful. However, as wild strains of Brucella have also been isolated from vaccinated animals, the authors recommend increasing the amount of vaccine to a full dose of 1 to 2 x 10(9) CFU and vaccinating young female animals between the ages of three and eight months. To avoid brucellosis in humans, people should be educated about the dangers of contact with infected animals and the consumption of raw milk and milk products.

Activin-A differentially regulates steroidogenesis by sheep granulosa cells.

Intra-ovarian factors, such as activin, are implicated in multiple aspects of follicular development in mammalian ovaries. This study was conducted to investigate a possible effect of activin-A on steroidogenesis in sheep granulosa cells in vitro. Sheep granulosa cells were obtained from medium antral follicles and cultured in a chemically defined RPMI -1640. Oestradiol and progesterone production, secreted by the cultured cells, was evaluated by enzyme-linked immunosorbent assay. In order to determine the dose effect of activin-A on steroidogenesis, granulosa cells were cultured in the presence of increasing concentrations of activin-A (0, 0.5, 5 and 50 ng ml(-1)) for 48 hours. The results revealed that activin-A exerts a differential effect on steroidogenesis in granulosa cells in such a way that it significantly (P < 0.05) suppressed progesterone production and enhanced oestradiol production. These results were confirmed by the time effect of activin-A on oestradiol and progesterone production in granulosa cells. In the absence of activin-A treatment, granulosa cells showed enhanced capacity to produce progesterone, but not oestradiol, as the time progressed from 12 to 48 hours. Treatment of sheep granulosa cells with 25 ng ml(-1)activin-A for 12, 24 and 48 hours significantly stimulated oestradiol production but inhibited progesterone production. These results suggest that activin-A is a local regulator of sheep folliculogenesis that might act to support differentiation in granulosa cells and suppress luteinisation.

Quantitative analysis of Op18 phosphorylation in childhood acute leukemia.

Oncoprotein 18 (Op18) is a major cystolic phosphoprotein constituent of leukemia cells. There is cumulative evidence that suggests a role for Op18 in integrating signals from diverse pathways involved in cell growth. Op18 phosphorylation is induced with proliferation in a variety of cell types, and is essential for cell cycle progression. In this study we analyzed the level of unphosphorylated Op18 and of its major phosphorylated forms, Op18a and Op18b, in a series of 177 childhood acute leukemias by means of quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Op18 phosphorylation was significantly correlated with the white blood count at the time of diganosis, and with a high percentage of cells in the S phase. Our findings suggest that strategies to inhibit Op18 expression or phosphorylation may be effective in inhibiting leukemia cell proliferation.

Equine babesiosis associated with strenuous exercise: clinical and pathological studies in Jordan.

Clinical, haematological and pathological studies were undertaken in Jordan in a stud of 103 racing horses clinically suffering from babesiosis and apparently healthy animals. Out of 47 horses which participated in strenuous exercise, three mares showed sudden onset of immobility and reluctance to move and two mares died. Clinical examination revealed that these five horses (group 1) had fever, anorexia, weakness and severe icterus and, in two mares, haemoglobinuria. Haematological examination revealed that all five horses were heavily parasitized with Babesia equi. This was also found in four horses (group 2) with no evidence of clinical babesiosis. In group 3 (94 horses), neither clinical signs nor B. equi were observed in the blood. The horses in group 1 and 2 recovered after treatment with imidocarb. When the mean values of white blood cell count, red blood cell count, haemoglobin and packed cell volume in group 1 were compared with those for groups 2 and 3, a significant difference was found (P < 0.05). A significant difference was also found when the mean values were compared before and after treatment. Examination of serum total protein, bilirubin and serum enzymes revealed a significant decrease in the mean value of total serum protein (P < 0.05), and a significant increase in the mean values of bilirubin (P < 0.05) in group 1 compared to groups 2 and 3. A significant elevation in the mean value of aspartate aminotransaminase, gamma-glutamyltransferase and creatine phosphokinase and a substantial elevation in the mean value of alkaline phosphatase was also observed in group 1 compared to groups 2 and 3. Postmortem examination of the dead horses showed that the animals had icterus, hepatomegaly and full urinary bladder with deep-red urine. Histopathological examination of the liver showed massive centrilobular degeneration and necrosis. The bile canaliculi and bile ducts were prominent and plugged with dark-brown to canary-coloured bile pigments. The lungs had congestion, oedema, and thrombosis of pulmonary veins. Our results suggest that the horses suffered from B. equal with clinical manifestation following exercise. The clinical, haematological and pathological findings indicate that the animals suffered from haemolytic anaemia which responded to imidocarb therapy.

Foetal anasarca in Awassi sheep.

Two anasarcous foetuses of Awassi sheep are described. The foetuses were removed from the dams by caesarean section because of dystocia due to failure of cervical dilation. Uterine incision was made in situ because uteri were so distended they could not be brought out from the site of incision. Large quantities of uterine fluids and abnormal thick placentas were found. One foetus weighed about 7 kg and the other 13 kg. The foetal heads were deformed: the upper jaw was prognathic and the left ear of the small foetus was cystic. Necropsy revealed subcutaneous musculature was soft and flabby and abdominal and thoracic cavities contained serosanguinous fluid. Histopathological examination revealed that only the larger foetus had focal aggregates of basophilic nucleated red blood cells and scattered megakaryocytes in the liver. We conclude that anasarca can occur in Awassi sheep, with and without associated extramedullary haematopoiesis.

A technique for identification of cellular proteins by western immunoblotting and immunoautoradiography of 2-D PAGE.

A major effort of our work has been devoted to the identification, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), of lymphoid polypeptides that are involved in lymphoid proliferation or differentiation. We have encountered problems during this effort pertaining to the exact localization of a polypeptide(s) in the silver stained gels that is recognized by western immunoblotting or proteins detected by autoradiography. In this paper, we present a method, using India ink stained/immuno-staining replica of 2-D gel nitrocellulose membrane (NCM) or India ink stained coupled with autoradiography in the case of phosphoproteins, which allows us to exactly localize the polypeptide spots detected by these methods in the silver stained 2-D PAGE. This method is expected to popularize and widen the use of 2-D PAGE technology in the investigation of cellular polypeptides.

National cross-sectional study of mastitis in dairy cattle in Jordan.

Between July 1991 and August 1992, 63 Jordanian dairy farms selected by stratified random sample were visited to identify the major causes and prevalence of intramammary infections in dairy cows. Of 773 cows examined 60% of all sampled quarters had > 283,000 cells/ml. The mean value of somatic cell count (SCC) was positively associated with age in lactations and negatively with herd size. Cows milked by bucket milking machines or in fully automatic parlours had a lower mean SCC than those milked by hand. Many management faults pertaining to milking procedure and maintenance of milking machines were noted. The most common isolate from clinical cases was Staphylococcus aureus (37.5%). Estimates of prevalence of bacterial pathogens in intramammary infections were: coagulase negative staphylococci (16.04%), S. aureus (9.41%), Klebsiella spp. (6.17%), Corynebacterium bovis (5.35%) and Brucella melitensis (4.52%). The results demonstrate the essential need for the development of a national mastitis control programme.

Hsp27 expression in neuroblastoma: correlation with disease stage.

BACKGROUND: The 27-kd heat shock protein (Hsp27) is differentially expressed in some malignancies, including breast carcinoma, leukemia, and malignant fibrous histiocytoma. In breast carcinoma, a high-level expression of Hsp27 has been associated with shorter disease-free survival in patients with localized disease. PURPOSE: We have observed variable levels of Hsp27 among neuroblastoma tumors. Our aim in this study was to investigate the relationship between Hsp27 expression and stage of the disease and N-myc gene copy number. METHODS: We determined Hsp27 protein levels in 53 neuroblastoma tumors representing different stages of the disease and in 17 neuroblastoma cell lines by quantitative two-dimensional polyacrylamide gel electrophoresis (PAGE). We also performed statistical analysis of Hsp27 levels in relation to stage of the disease and to N-myc gene copy number. RESULTS: Increased Hsp27 expression in neuroblastomas was associated with limited stage disease and inversely correlated with N-myc gene amplification, a feature known to predict poor clinical outcome. An inverse correlation was also observed between N-myc gene amplification and Hsp27 protein levels among the neuroblastoma cell lines analyzed. Immunohistochemical staining of sections of neuroblastomas showed that Hsp27 was most prominently expressed in the cytoplasm of large ganglionic tumor cells present in neuronally differentiated areas of the tumors. Induction of neuronal differentiation in SMS-KCNR neuroblastoma cells using retinoic acid resulted in an increase in Hsp27. CONCLUSION: High level expression of Hsp27 in neuroblastoma is a feature of limited stage, differentiated tumors. IMPLICATION: Hsp27 may play a part in the biology of neuroblastomas with a favorable outcome.

Data base analysis of protein expression patterns during T-cell ontogeny and activation.

We have developed a data base of lymphoid proteins detectable by two-dimensional polyacrylamide gel electrophoresis. The data base contains two-dimensional patterns and derived information pertaining to polypeptide constituents of unstimulated and stimulated mature T cells and immature thymocytes, single-cell-derived T- and B-cell clones, leukemia cells, and lymphoid cell lines. Using this data base, we have compared the protein constituents of mature T cells and immature thymocytes before and after mitotic stimulation. A subset of polypeptides that are induced in mature T cells following mitotic stimulation were found to be constitutively expressed in immature thymocytes. Other polypeptides exhibited differences in their expression between mature and immature thymocytes in a manner unrelated to proliferation. The identity of several constitutively expressed or mitotically induced proteins in lymphoid cells was established by microsequencing. These initial findings point to significant differences in the molecular pathways leading to proliferation between mature and immature T cells. The construction of this database should facilitate further studies of lymphoid differentiation and function.

PCNA levels in neuroblastoma are increased in tumors with an amplified N-myc gene and in metastatic stage tumors.

N-myc oncogene amplification in neuroblastoma has been found to be significantly associated with advanced stage disease and tumor progression. However, there is a lack of data on tumors, regarding the relationship between N-myc gene amplification and proliferation activity. Proliferating cell nuclear antigen (PCNA) is a proliferation-induced 36 kD nuclear protein that is the auxiliary component of DNA polymerase delta. PCNA levels in tissues have been found to correlate with proliferative activity. We have examined PCNA levels in neuroblastomas in relation to N-myc gene amplification and tumor stage. Statistically, significantly higher levels of PCNA were observed in tumors with an amplified N-myc gene relative to tumors with a single gene copy. The highest levels of PCNA were observed in advanced stage tumors with an amplified N-myc gene. Treatment of neuroblastoma cells in culture with retinoic acid, which induces differentiation, resulted in a substantial decrease in PCNA. Our results suggest that PCNA levels may reflect differences in proliferative activity between neuroblastomas, related to stage of the disease and to N-myc gene copy number.

Activation of resting peripheral blood lymphocytes through the T cell receptor induces rapid phosphorylation of Op18.

Op18 is a highly conserved major cytosolic phosphoprotein that has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. After mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study, we have examined the phosphorylation of Op18 in freshly isolated PBL after activation of the T cell receptor by OKT3. Quantitative analysis of Op18 phosphorylation was undertaken by metabolic labeling with 32Pi and PhosphorImager analysis of two-dimensional gels. After 10 or 15 min of activation by OKT3, one of the three major phosphorylated forms of Op18, designated Op18c, increased approximately 10-fold, which represented a most pronounced change among a large number of phosphoproteins analyzed. In time course experiments, increased Op18 phosphorylation to yield Op18c was observed as early as 2 min. Continued OKT3-induced activation for 20 to 72 h resulted in a further increase in phosphorylated Op18 forms, which paralleled new Op18 synthesis and occurred at a time when cells were entering S-phase, as determined by [3H]-thymidine incorporation. Inhibitors of lymphoid proliferation, cyclosporin A and RPM, had no effect on early (less than 15 min) phosphorylation. Addition of calphostin C, a specific inhibitor of protein kinase C, 1 min prior to stimulation of resting T cells with OKT3 completely inhibited further phosphorylation of Op18. Incubation of PBL with calphostin C for 75 min decreased constitutive levels of phosphorylated Op18. In contrast, inhibition of cyclic nucleotide-dependent protein kinases with HA1004 had no effect on Op18 phosphorylation. Activation of cAMP-dependent protein kinase with Forskolin or 8Br-cAMP did not increase Op18 phosphorylation. Our results suggest that Op18 phosphorylation is mediated by protein kinase C activation as an early event in T cell activation through the T cell receptor.

Proliferation-related expression of p19/nm23 nucleoside diphosphate kinase.

High level expression of the nm23-H1 gene, which encodes for a nucleoside diphosphate kinase, has been found to correlate with diminished metastasis in some tumors but not in others. We have previously identified the protein product of the nm23-H1 gene in two-dimensional electrophoretic gels and have designated it p19/nm23. In neuroblastoma, higher levels of p19/nm23, which are associated with amplification of the N-myc oncogene, large tumor mass, and metastasis, were observed in advanced stage tumors compared with limited stage disease. Because of the variable expression of nm23-H1 in different tumors, we have investigated the relationship between amounts of the protein and cell proliferation. The levels of p19/nm23 were compared between resting and mitotically stimulated normal human PBLs and in leukemia cells. The amount of p19/nm23 increased in normal lymphocytes in response to mitotic stimulation and paralleled the increase in DNA synthesis. In leukemia cells obtained from patients with different subtypes of acute leukemia, p19/nm23 levels were also increased relative to resting normal lymphocytes. Treatment of mitotically stimulated lymphocytes with cyclosporin, which inhibits proliferation, blocked the increase in p19/nm23; treatment of the leukemia cell line HL-60 with dimethylsulfoxide, which induces terminal differentiation, resulted in diminished levels of p19/nm23. Our data therefore provide evidence that nm23-H1 expression is related to cell proliferative activity.

Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin.

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.

Involvement of OP18 in cell proliferation.

Op18 is a highly conserved major cytosolic phosphoprotein that is expressed at high levels in acute leukemia and in neuroblastoma. In this study we present evidence pointing to a role for Op18 in cellular proliferation. Blocking of Op18 mRNA translation using antisense oligonucleotides delayed entrance of mitotically stimulated normal peripheral blood lymphocytes into the S phase. Moreover treatment of HL-60 promyelocytic leukemia cells with DMSO or PMA which induced terminal differentiation resulted in a decrease in the level of Op18 RNA and protein. Inhibition of lymphoid proliferation with cyclosporin also resulted in reduced Op18 levels.

Characterization of the gene for a proliferation-related phosphoprotein (oncoprotein 18) expressed in high amounts in acute leukemia.

The oncoprotein 18 (Op18) gene encodes a proliferation-related cytosolic phosphoprotein, which is induced in normal lymphocytes following mitogenic stimulation. Studies of the Op18 gene are of particular interest because of the proposed role of Op18 protein in signal transduction and because of its occurrence in markedly increased amounts in acute leukemia cells. We have recently reported the cloning and sequencing of two cDNA clones for Op18 (1 and 1.5 kilobases). Both clones code for the same 149-amino acid polypeptide; however, they differ in their 3'-region as a result of alternative polyadenylation. We report here the sequencing of the Op18 gene and describe its expression in leukemia. The Op18 gene, which is 6.3 kilobases in length, is comprised of five exons and four introns and exhibits features that are common to other genes involved in cellular growth and proliferation. The increase in Op18 polypeptide in leukemia is associated with increased RNA transcription without gene amplification or rearrangement. Treatment of K562 leukemia cell line with hemin that induces terminal differentiation resulted in decreased expression of Op18. Our findings suggest that the high amount of Op18 protein in acute leukemia results from increased expression of a structurally unaltered gene.

High levels of p19/nm23 protein in neuroblastoma are associated with advanced stage disease and with N-myc gene amplification.

The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.

Highly resolving two-dimensional gels for protein sequencing.

Two-dimensional (2D) PAGE, using carrier ampholytes for the first-dimension separation, has provided a tool for the simultaneous analysis of cellular proteins. To extend the utility of 2D PAGE to the preparative level, we have investigated the use of immobilized pH gradients (IPG) for the first-dimension separation. The results we have obtained indicate that as much as 1 mg of cellular protein can be loaded onto a single IPG gel without loss of resolution. Mutant polypeptides previously detected in carrier ampholyte-based 2D gels were equally detectable in IPG-based 2D gels. With IPG gels several hundred cellular polypeptides can be isolated, from as few as 10 gels, in sufficient amount for sequencing with current sequencing technology. We therefore conclude that IPG greatly enhances the prospects for the large-scale sequencing of cellular proteins for the development of 2D gel-related protein data bases and for the identification of new polypeptide gene products, with the attendant implications for a genome sequencing effort.