Evaluation and comparison of cytotoxicity and bioactivity of chemomechanical caries removal agents on stem cells from human exfoliated deciduous teeth.

AIM: To investigate and compare the cytotoxicity and bioactivity of CMCR agents on stem cells derived from exfoliated deciduous teeth. METHODOLOGY: MTT assay, flow cytometry, Alizarin Red staining and scratch assay were used to assess the cellular viability, apoptosis, calcium matrix deposits and cell migration, respectively. The gene expression of ALP and BMP-2 was measured with RT-PCR. One-way ANOVA and Bonferroni post-test was used for statistical analysis. RESULTS: 0.5% Carisolv showed highest cell proliferation and calcium matrix formation, whereas 0.5% Papacarie reported the highest% live cells and cell migration. The highest mRNA expression of ALP and BMP-2 was reported in SHEDs cultured in 0.5% Papacarie (after 72 h incubation) and 0.5% Carisolv (after 24 h incubation), respectively. CONCLUSION: CMCR agents are biocompatible and bioactive when cultured in stem cells derived from exfoliated primary teeth.

Phenotypic Prenatal Diagnosis of Chronic Granulomatous Disease: A Useful Tool in The Absence Of Molecular Diagnosis.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency disorder affecting the microbicidal function of the phagocytes. It is characterized by susceptibility to recurrent infections leading to significant morbidity and mortality. Antibacterial and antifungal prophylaxis, though, has significantly reduced the rate and severity of the infections; the breakthrough infections still remain a challenge. Currently, allogenic haematopoietic stem cell transplantation is the only curative option which is very expensive and unavailable for many due to lack of suitable donor. Thus, prenatal diagnosis (PND) forms an important component of management in the affected families. PND is challenging in families approaching late in pregnancy with an uncharacterized molecular defect. In such cases, PND can be performed by analysis of NADPH activity of fetal blood (FB) neutrophils at 18-20 weeks of gestation. Cord blood samples at 18 weeks of gestation from healthy control were used to establish normal ranges for NBT and DHR. PND was offered for six pregnancies (NBT: n = 3, DHR: n = 6) with index cases of CGD confirmed by abnormal NBT and DHR analysis. NBT and DHR tests were found to be negative for all the six cases, confirming the same on samples post-delivery. NBT and DHR tests offer a rapid and sensitive PND of CGD in the absence of facilities for molecular diagnosis. It was observed that addition of CD15 along with CD45 led to an accurate DHR analysis. It is recommended to perform the diagnosis with adequate precautions only at centres with considerable experience and expertise in the diagnosis of CGD.

Hyperimmunoglobulin syndrome due to CD40 deficiency: possibly the first case from India.

Hyperimmunoglobulin M (HIGM) type 3 due to CD40 deficiency is a very rare syndrome. Only 16 cases have been reported thus far. The clinical presentation is very variable. We present the first case of this rare disorder from India. The case is of a two-and-a-half-year-old female, with a history of repeated episodes of skin infections and diarrhea since birth. Laboratory evaluation revealed elevated absolute lymphocyte count and an absolute neutrophil count (ANC) of 1026/mm3. The lymphocyte subset analysis showed normal absolute counts of Natural Killer (NK) cells and elevated absolute counts of T-cells (CD4 and CD8) and B-cells. The serum immunoglobulin estimation showed low levels of IgG, IgA, IgE and an elevated level of IgM. The CD154 analysis was normal and expression of CD40 was absent on the B-cells. Molecular analysis showed a novel mutation, with deletion of 3bp (AAG) [p.Glu107GlyfsX84] in the homozygous state, in the CD40 gene. Thus the patient was diagnosed as HIGM type 3. The parents were screened and counseled regarding prenatal diagnosis at the time of next pregnancy.

Diagnostic approach to primary immunodeficiency disorders.

Primary immunodeficiency disorders (PIDs) are a heterogeneous group of inherited disorders that affect different components of the immune system. There are more than 150 different disorders which have been described till date. Despite major advances in the molecular characterization of PIDs over the last 20 years, many patients remain undiagnosed or are diagnosed too late with severe consequences. Recognizing different clinical manifestations of PID is the first most important step. It should be followed by use of appropriate diagnostic tools from a vast number of investigations available. This review will focus on important presenting features of PID and laboratory approach for diagnosis of suspected cases of PID.

Clinical profile of leukocyte adhesion deficiency type I.

Leukocyte adhesion deficiency type I (LAD-I) is a rare, inherited immunodeficiency with defect in the recruitment of leukocyte to the site of inflammation. Patients with severe LAD-I have absent or markedly reduced expression of CD18 and CD11. Here we report clinical profile of 7 cases of LAD-I diagnosed at our center over a period of 3 years. Recurrent skin and mucous membrane infections were the major presenting manifestations. All children had a history of delayed cord separation.

Three way translocation in a new variant of t(8;21) acute myeloid leukemia involving Xp22.

The t(8;21)(q22;q22) is one of the most frequent chromosomal abnormality associated with acute myeloid leukemia (AML) M2 sub type. The additional chromosomal abnormalities including structural and numerical are frequently reported with the translocation, t (8;21)(q22;q22). We report a case of AML-M2 with t(X;8;21)(p22;q22;q22) associated with loss of Y chromosome. Using a dual color fluorescence in situ hybridization (FISH) analysis with ETO and AML1 probes, we demonstrated an ETO/AML1 fusion signal on the derivative chromosome 8 and one ETO signal on derivative Chromosome Xp22. The patient did not respond to therapy and follow-up of cytogenetics revealed same chromosome abnormality. Hence, this three way translocation involving X chromosome might be associated with poor prognosis.

Optimising methods of red cell sedimentation from cord blood to maximise nucleated cell recovery prior to cryopreservation.

Human cord blood is now an established source of stem cells for haematopoietic reconstitution. Red blood cell (RBC) depletion is required to reduce the cord blood unit volume for commercial banking. Red cell sedimentation using hydroxy ethyl starch (HES) is a standard procedure in most cord blood banks. However, while standardising the procedure for cord blood banking, a significant loss of nucleated cells (NC) may be encountered during standard HES sedimentation protocols. This study compares four procedures for cord blood processing to obtain optimal yield of nucleated cells. Gelatin, dextran, 6% HES and 6% HES with an equal volume of phosphate-buffered saline (PBS) were compared for RBC depletion and NC recovery. Dilution of the cord blood unit with an equal volume of PBS prior to sedimentation with HES resulted in maximum NC recovery (99% [99.5 +/- 1.3%]). Although standard procedures using 6% HES are well established in Western countries, they may not be applicable in India, as a variety of factors that can affect RBC sedimentation (e.g., iron deficiency, hypoalbuminaemia, thalassaemia trait, etc.) may reduce RBC sedimentation and thus reduce NC recovery. While diluting cord blood with an equal volume of PBS is a simple method to improve the NC recovery, it does involve an additional processing step.

Intracranial haemorrhage in severe haemophilia: prevalence and outcome in a developing country.

Intracranial haemorrhage (ICH) is a common cause of morbidity and mortality in haemophilic patients all over the world. From 1995 to 2004, we have investigated 37 patients with 43 episodes of ICH at our Comprehensive Haemophilia Care Center from a total of 600 registered patients. Diagnosis of ICH in the patients was confirmed by clinical, haematological and computed tomographic imaging data. Three patients died despite replacement therapy while one child who had a ventriculo-atrial shunt for acute hydrocephalus also died before further intervention. One of the four patients who died also had severe aplastic anaemia for 6 years in addition to severe haemophilia. Detailed history obtained from 143 families with haemophilia attending the Genetic Diagnosis Clinic at our Center showed a positive history of cerebral bleed in 39 episodes in 37 patients. Sixteen families gave a history of death in the family of haemophilic patients due to ICH, while in the remaining 21 families, the patients had survived the episode after treatment elsewhere. However, the ICH was not confirmed by image data in these cases. The treatment protocols were also not available in these cases. Conservative factor replacement therapy 100% correction for 3 days followed by 50-60% correction for 7 days) coupled with the epsilon amino caproic acid, the antifibrinolytic agent at least for 30 days led to a mortality (10.8%) similar to that of the western countries and almost no morbidity. Surgery was not required in any of these patients except in one elderly patient with HIV infection on antiretroviral therapy.

First-time development of FVIII inhibitor in haemophilia patients during the postoperative period.

Development of inhibitor to FVIII in haemophilia patients is well-known and is not uncommon. However, their development for the first time during the postoperative period has hardly been reported. In a developing country such as India, where resources are limited, development of such an eventuality may prove disastrous. However, as many of our patients are sparingly treated, therefore, even if they test negative for the inhibitor preoperatively, they may get the requisite FVIII antigenic stimulation during the preoperative and immediate postoperative period, leading to the development of inhibitors during this critical time of wound healing. We describe here six patients who developed such an inhibitor, from a group of 35 patients with haemophilia A who underwent various surgical procedures (19%). We stress that such an eventuality may not remain rare in developing countries as more patients of severe haemophilia undergo surgery and are therefore challenged for the first time in their life with large amounts of FVIII concentrate during their preoperative period.

Low cost autologous peripheral blood stem cell transplantation performed in a municipal hospital for a patient with plasma cell leukaemia.

Autologous peripheral blood stem cell transplantation (PBSCT) is a costly procedure. In India, the cost varies from US$20000 to 25000 and most patients cannot afford it. Using several cost-cutting measures, we were able to treat a patient with plasma cell leukaemia by autologous PBSCT. A 42-year-old-male presented with plasma cell leukaemia. He was treated with VAD therapy, followed by high-dose cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) for mobilization of peripheral blood stem cells. The patient was conditioned with high dose melphalan, followed by autologous PBSCT. The procedure was performed in a municipal hospital in which there was no prior experience with stem cell transplantation. Costs were reduced by: (i) using oral medication whenever possible; (ii) having a relative of the patient prepare his food under medical guidance; (iii) starting G-CSF on day 7 rather than on day 1; (iv) short-term storage of the PBSC in an ordinary refrigerator at 4 degrees C without cryopreservation; (v) infusing a large number of CD34+ cells, which shortened the time to engraftment; (vi) delegating many of the functions of a marrow transplant nurse to a resident physician. The cost of transplantation was thereby reduced to about US$ 6000, with successful engraftment by day +13. The patient remained in remission for 7 months, after which he relapsed and was treated with chemotherapy and electron beam radiation to the skin.

Systemic capillary leak syndrome preceding plasma cell leukaemia.

We report a patient with plasma cell leukaemia with systemic capillary leak syndrome, a rare disorder often associated with monoclonal gammopathy. In this patient, the manifestation of capillary leak syndrome antedated the diagnosis of plasma cell leukaemia by 5-6 months. During that time, he was repeatedly admitted to the hospital with weight gain, congestive cardiac failure, cough and anasarca in the presence of normal renal function, liver function and normal echocardiography. On presentation, a serum protein electrophoresis showed monoclonal IgG; the blood smear showed 60% plasma cells with a total count of 4.4 x 10(9)/l. A bone marrow aspirate showed replacement of the normal marrow by sheets of immature plasma cells. His systemic capillary leak syndrome initially responded to decongestive therapy with terbutaline and aminophylline but later on he became refractory to them and responded to vincristine, doxorubicin and dexamethasone (VAD) combination therapy only transiently. Danocrine and pentoxifylline, added during VAD chemotherapy, did not produce a durable response in capillary leak syndrome, which finally responded to autologous peripheral blood stem cell transplantation (PBSCT). After PBSCT, he remained free of capillary leak for 10 months without terbutaline, pentoxifylline corticosteroids, aminophylline or danocrine. His disease relapsed without recurrence of the capillary leak. He died 15 months after PBSCT and 20 months after the diagnosis of plasma cell leukaemia.

Ophthalmic surgery in haemophilia.

A wide range of ophthalmic surgical procedures were conducted in five patients with haemophilia of varying severity (one severe and four mild) aged between 8 and 75 years. The operations included intraocular lens implantation, trabeculectomy and vitrectomy. Successful postoperative outcome with haemostasis was achieved in all patients with moderate use of clotting factor concentrates (3000-7000 IU), simultaneous use of oral epsilon amino caproic acid therapy and intravenous deamino-8-D-arginine vasopressin (desmopressin; DDAVP) wherever feasible. None of the patients had circulating inhibitor. One of the patients with milder disease (FVIII 32%) was referred to us after he was operated on for hyphaema elsewhere, without prior knowledge of his diagnosis of haemophilia. Thus, satisfactory eye surgery in patients with haemophilia is possible with a restricted amount of factor concentrates with gratifying results.

Venous thromboembolism in young patients from western India: a study.

The goal of this article is to study the association of known markers of thrombophilia with venous thrombosis in young patients (< 45 years) from the Western part of India. A prospective study of 432 patients (252 males and 180 females, age 1-45 years) was conducted between 1994 and 2000 (6 years). The diagnosis was confirmed in all the patients by ultrasound with Doppler or by a computed tomograph (CT) scan of the brain with or without contrast depending on the case. Detailed clinical examination, and family history was taken to establish recurrent thrombosis and familial occurrence of thrombosis. The markers studied were protein C, protein S, antithrombin (AT) III, factor V Leiden mutation, prothrombin gene G20210A polymorphism, and the thermolabile MTHFR variant C677T polymorphism, using appropriate techniques. Lupus inhibitor was tested in the first 72 patients using Dilute Russel Viper Venom Time (DRVVT) test, and anticardiolipin antibodies were tested by enzyme-linked immunosorbent assay. Protein C, protein S, and AT III deficiency was detected in 9.5%, 6.5%, and 2.6%, respectively, among the patients. Anticardiolipin antibody was present in 9.9% of the patients, whereas lupus anticoagulant was present in 8.3% of patients; factor V Leiden mutation was detected in 3% of patients; thermolabile variant of MTHFR C677T polymorphism was present in 14.9% of patients with 1.2% homozygotes. Prothrombin G20210A polymorphism was not detected in any sample in this population. One hundred and four patients of 432 (24.9%) had recurrent attacks of thrombosis without any proximate precipitating cause, whereas 7.5 % of the patients had another close member of the family with a history of deep venous thrombosis. Eighty-six members from 28 families (out of 32 families giving family history of thrombosis) were investigated and found to have protein C and protein S deficiency in seven each; factor V Leiden was present in 6, and MTHFR C677T polymorphism was present in 5 cases. Hence, 25 of 86 members (28%) from the family of patients with familial history deep venous thrombosis had positive markers for thrombophilia. Thus, we could show that in young patients presenting with thrombosis, at least 34% of them had a demonstrable cause for thrombophilia. Prothrombin gene polymorphism G20210A seems to be nonexistent in our population and AT III deficiency also appears to be low compared to other markers of thrombophilia. There is a high prevalence of variant MTHFR C677T in our series, but the incidence of MTHFR C677T in our general population is also high. Hence, the significance of this finding in our cases of deep venous thrombosis remains to be seen, but we did not see any homozygotes when we tested 70 randomly selected asymptomatic persons, whereas in the present series, 1.8% of the patients had homozygosity for the MTHFR C677T polymorphism.

Transfusion transmitted diseases in haemophilics from western India.

BACKGROUND & OBJECTIVES: Transfusion related human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) infections have been a major cause for morbidity and mortality in the haemophilic population in the west. The prevalence of these markers of transfusion transmitted viral diseases in severe and moderate haemophilia patients was studied. METHODS: The seropositivity for these viral markers was evaluated in 400 haemophilics (323 severe and 77 moderate) in a 5-year survey starting from 1995. First 188 of these patients were also tested for HCV. Serological tests for HIV, HBsAg and HCV were done by third generation ELISA; positive samples were also confirmed by Western blot. RESULTS: Fifteen of the 400 patients were found to be HIV positive (3.8%), 24/400 were HBsAg positive (6%) and 45/188 (23.9%) were positive for HCV (28 for both non-structural and core antigen, 13 for core only and 4 for non-structural antigen only). The lowest age of HIV positivity was 12 yr and that of HCV positivity was 8 yr. INTERPRETATION & CONCLUSION: The above study shows a reduction in blood product related HIV transmission in severe and moderately affected haemophilics but more stringent policy for blood product usage, universal hepatitis C screening, hepatitis B vaccination and continuous awareness programmes for medical staff, general public and patients is needed to reduce the incidence of these diseases in haemophilics.

Combined factor V and VIII deficiency in Indian population.

The clinical and haematological heterogeneity in cases of the rare combined factor V and VIII deficiency has not been reported so far from India. Nine such cases belonging to five unrelated families have been analysed in the present study for the various haematological and clinical parameters. A very mild clinical presentation is seen in all these cases. The clinical manifestations, however, do not correlate with the plasma levels of these factors.