Biomedical subjects
S K Singhal
Publications and source records attributed to S K Singhal.
Development and validation of a stability indicating HPLC assay method for cyclosporine in cyclosporine oral solution USP.
The suitability of the United States Pharmacopeia (USP) assay method for analysing stressed samples of cyclosporine oral solution USP was evaluated for stability samples by analyzing cyclosporine oral solution after acid, alkali, hydrogen peroxide, heat and light treatment. Some of the degradants generated during stress testing, as well as dihydrocyclosporine A, which is a known degradant of cyclosporine A, were not adequately resolved from the cyclosporine peak and mobile phase adjustments did not improve the resolution. In addition, isocyclosporine A, another known degradant of cyclosporine, could not be quantitated as it was eluting too early with the system peaks. Therefore, a binary gradient, reverse phase, stability indicating, HPLC method for the assay of cyclosporine in cyclosporine oral solution USP has been developed and validated. Analysis of degraded samples showed that the cyclosporine A eluted as a spectrally pure peak resolved from its degradation products.
CXCR1 and CXCR2 are rapidly down-modulated by bacterial endotoxin through a unique agonist-independent, tyrosine kinase-dependent mechanism.
The expression of the seven-transmembrane domain chemokine receptors CXCR1 and CXCR2 modulates neutrophil responsiveness to the chemoattractant IL-8 and a number of closely related CXC chemokines. In the present study, we investigated the mechanism by which bacterial LPS induces the down-modulation of IL-8 responsiveness and CXCR1 and CXCR2 expression on human neutrophils. Treating neutrophils with LPS reduced IL-8R expression to 55 +/- 5% of the control within 30 min and to 23 +/- 2% within 1 h of stimulation. Furthermore, this down-modulation could not be attributed to increased concentrations of IL-8, TNF-alpha, or IL-1beta, since ELISA studies indicated that LPS-stimulated neutrophils did not release detectable amounts of these proteins before 2 h poststimulation. The tyrosine kinase (TK) inhibitors genistein and herbimycin A attenuated the LPS-mediated down-modulation of CXCR1 and CXCR2, indicating that the activation of a TK is required for LPS to mediate its effect. The effect of LPS on receptor expression paralleled the hyperphosphorylation of the protein TK p72syk. Although IL-8 induced a comparable down-modulation of CXCR1 and CXCR2, TK inhibitors did not attenuate this effect. These studies provide the first evidence of an agonist-independent, TK-dependent pathway of chemokine receptor regulation by endotoxin.
Management of congenital lobar emphysema with endobronchial intubation and controlled ventilation.
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Successful pregnancy in a patient with previous bladder exstrophy.
Bladder exstrophy is an unusual congenital anomaly. Patients becoming pregnant with such anomaly after surgical repair are even rarer. The present case reports a lower segment Caesarean section delivery of a living healthy baby in a woman who had been operated on for bladder exstrophy at the age of 18 years. This is the first case of this type seen in our institution in 35 years.
Uterus didelphys, a rare cause for tubal sterilization failure.
Uterus didelphys is a rare congenital anomaly. It can result in a variety of misadventures even in the hands of an expert. The present case reports uterus didelphys as the cause for tubal sterilization failure. This is the first case of this type seen in our institution in 35 years.
Suppression of myelopoiesis and myeloid leukemia cell line proliferation by a novel bone marrow-derived factor, reptimed.
Bone marrow is the major site of hematopoiesis in the adult mammal. Bone marrow contains a highly organized microenvironment for the support of hematopoietic stem and progenitor cells, including the production of growth factors. Bone marrow cells also produce negative regulatory factors which may regulate hematopoiesis and inflammatory responses. In this paper we describe Reptimed, a unique bone marrow-derived factor with inhibitory activity for myelopoiesis and in vitro growth of myeloid cell lines. Reptimed was partially purified from bone marrow supernatants using a combination of solid-phase extraction and size exclusion chromatography. Reptimed is < 1000 Da MW and is water soluble. Reptimed inhibited growth of granulocyte-macrophage and macrophage colonies as well as proliferation of several myeloid leukemia cell lines. Reptimed may be part of a hemoregulatory circuit.
Pleomorphic adenoma presenting as a base of tongue mass.
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Nonunion of uterine incision: a rare cause of secondary postpartum haemorrhage: a report of 2 cases.
Secondary postpartum haemorrhage after Caesarean section due to non-union of the uterine incision is an unusual complication. Two women who had had lower segment Caesarean section for placenta praevia presented after 2 and 3 weeks respectively with shock due to massive postpartum haemorrhage. The uterine scars had not healed in both of these women who were managed by emergency subtotal hysterectomy.
Prevention and reversal of renal allograft rejection by antibody against CD45RB.
Rejection continues to be the single largest impediment to successful organ transplantation. Antilymphocyte globulin, which contains antibodies that react with the leukocyte common antigen known as CD45, has proved to be one of the most effective agents for preventing rejection. We have shown earlier that a monoclonal antibody directed against the RB isoform of CD45 substantially inhibits the alloreactivity of human CD4+ lymphocytes in vitro. Here we investigate whether CD45RB could be an appropriate target for preventing renal allograft rejection in mice. Mice treated with two injections of a monoclonal antibody (MB23G2) raised against CD45RB protein all survived and had normal renal function. Furthermore, this antibody reversed acute rejection when therapy was delayed until day 4, and the mice survived for their natural lifespan. The immunosuppression achieved may find application in the prevention and treatment of transplant rejection in man.
Therapy for mouse renal allograft rejection by monoclonal antibody to CD45RB.
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Effect of oil treatments on the levels of antinutritional factors in Callosobruchus chinensis (L.) infested stored pulses.
Coconut, groundnut and mustard oil treated whole red gram, green gram and bengal gram were infested with Callosobruchus chinensis (L.), stored for 6 months, and observed at monthly intervals for antinutritional factors (phytic acid, trypsin inhibitor activity (TIA) and saponins). The values of phytic acid and TIA of treated and untreated pulses were similar at month one. The saponin content of green gram and Bengal gram were, however, higher in control than the oil treated pulses at month one. From month two to six the untreated controls had progressively more antinutrients than their oil treated counterparts. In mustard and groundnut oil treated pulses, the antinutrients remained static for 6 months, however, coconut oil treated pulse had increased antinutrients from end of month four onwards. The storage period was associated with the level of insect infestation which in turn influenced the antinutrient contents of pulses.
Separation of allostimulatory and natural suppressor/stem cell functions of murine bone marrow--implications for bone marrow transplantation.
Natural suppressor (NS) activity is defined by the ability of a null cell population to inhibit antigenic and mitogenic activation of lymphoid cells. We had previously demonstrated that murine and human bone marrow was a rich source of NS activity and could be enriched in a population of large, low-density cells after counterflow centrifugal elutriation (CCE). In this report we confirm previous findings in the mixed lymphocyte reaction (MLR) that NS activity is an endogenous function of BM and is not present in peripheral immune tissues. Furthermore, suppression by BM NS cells in the MLR is not MHC restricted, which distinguishes NS activity from veto activity also associated with BM. To enrich for NS activity, BM cells were separated into 3 fractions according to size and density by CCE. Fraction (F) 1 contained small, high-density cells; F2 contained cells of intermediate size and density; and F3 comprised large, low-density cells. Addition of CCE-enriched fractions to the MLR revealed potent NS activity associated with F3, consistent with our previous findings in the Ab response. CCE-enriched fractions of BM cells were also used as stimulators in an MLR to determine which fraction, if any, could support alloantigen-induced proliferation. The use of unfractionated C57Bl/6 (H-2b) BM cells as stimulators for BALB/c (H-2d) splenic responders resulted in little detectable proliferation as compared with that induced by C57Bl/6 splenic stimulators. However, when elutriated fractions were used as stimulators, F1 BM cells induced a significant proliferative response, albeit to a lesser magnitude than spleen cells. In order to determine which fraction contained hematopoietic progenitors, CCE-separated cells were assayed for granulocyte/macrophage colony formation (CFU-GM). F3 BM was enriched five-fold for CFU-GM progenitors as compared with unfractionated BM, while progenitors were virtually absent in F1 and F2 BM. The results suggest that CCE may represent an effective way of removing potential alloantigen-presenting cells (F1) from BM, while retaining hematopoietic progenitors and NS activity (F3). The implications of the findings as they relate to allogeneic BM transplantation are discussed.
Suppression of mixed lymphocyte reactivity by murine bone-marrow-derived suppressor factor--inhibition of proliferation due to a deficit in IL-2 production.
Murine bone marrow natural suppressor cells constitutively produce a soluble factor, BDSF, that suppresses murine Ab responses in vitro. BDSF is recovered in supernatants of cultured BM cells and resides in an MW range between 1-10 kD. We now demonstrate that BDSF suppresses proliferation in the mixed lymphocyte reaction (MLR). In order to elucidate the potential mechanism of action, BDSF was tested for the ability to interfere with IL-2-mediated T cell proliferation. BDSF was not able to inhibit IL-2-driven proliferation of the cell lines CTLL-2 and HT-2. However, examination of MLR culture supernatants revealed that suppression of proliferation by BDSF was associated with decreased levels of IL-2 production. Furthermore, responsiveness of BDSF-treated cultures could be reconstituted by adding exogenous IL-2, at the time of plating. The results suggest that hyporesponsiveness in the presence of BDSF is due to a deficit in IL-2 production, preventing expansion of Ag-stimulated cells.
Further characterization of murine bone marrow-derived natural suppressor cells. Potential relationships between NS and NK/LAK activities.
Murine bone marrow (BM) cells regulate a variety of immune responses via an endogenous natural suppressor (NS) activity. We demonstrate that BM-derived NS activity resides in an enriched fraction of large, low-density cells which have a high proliferative rate. Complement-dependent lysis of BM cells by antibody directed against markers of Veto and NK/LAK cells had no effect on NS activity. The BM of SCID mice and their littermate C.B-17 possessed normal NS activity. Conversely, the BM of NK-deficient C57 beige mice displayed reduced NS activity as compared to normal C57 black mice. Long-term BM cultures (LTBMC) generated in medium containing supernatants of Con A-stimulated (CAS) rat spleen cells resulted in the emergence of a population of cells which possessed NS activity greater than that of fresh BM cells. The LTBMC were also potent effectors of NK activity, as compared to fresh BM, which had little NK activity. Thus, while NS, NK/LAK, and Veto cells are all nonspecific effectors of immune suppression, the exact relationship between them is not clear.
Serum lipids in healthy tribals living at high altitudes in the Himalayas.
Serum lipids of 70 healthy volunteer tribals (36 males, 34 females) living at high altitudes (3100-4500 metres) in the Himalayas were studied. The mean total cholesterol, triglycerides, HDLc, LDLc and VLDLc levels for the group were 178.6 +/- 19.5, 101.4 +/- 18.3, 44.1 +/- 12.4, 114.5 +/- 17.3 and 20.2 +/- 3.6 mg/dl respectively. There was no significant difference between the two sexes and the various age groups. The total cholesterol/HDL ratio for men and women was 4.2 and 3.9 respectively. The various interacting factors responsible for overall pattern of lipids in these individuals is discussed.
Identification of a human bone marrow-derived immunoenhancing factor, BDEF.
In this report we describe the production and biological activity of human bone marrow-derived enhancing factor (BDEF). This factor is the constitutive product of cultured human BMC and could initially be recovered by ultrafiltration of cell-free BM supernatants to yield a crude fraction of Mr greater than 10,000 Da. This preparation can enhance the Ab response of human tonsillar cells, as well as murine spleen cells, to SRBC. HPLC fractionation of BM supernatants enriches for enhancing activity in a peak with an approximate Mr of 60 kDa. PAGE gel analysis reveals two protein bands which migrate to this area, one of 60 kDa, and a slightly smaller protein at 55 kDa. Antibodies generated against the above two proteins were shown to be specific by Western blotting and could recognize the native BM proteins as determined by ELISA. The antibodies were used to affinity purify the respective proteins, p60 and p55. The BM protein p60, but not p55, was able to enhance Ab synthesis in vitro and was also mitogenic for murine BMC and thymocytes. The addition of anti-p60 Ab to human tonsillar cells cultured with SRBC and human BDEF preparations resulted in abrogation of enhancement. These findings support the notion that the BM protein p60 is BDEF and that it may represent a novel enhancing molecule produced by normal human BM.