Características hematológicas y bioquímicas en pacientes con y sin diabetes mellitus tipo 2 (DM2) sometidos a hemodiálisis durante un año de seguimiento / Hematologic and biochemical characteristics in patients with and without type 2 diabetes mellitus undergoing hemodialysis during a one-year follow-up period

Objetivo: Reportar si existen diferencias hematológicas y bioquímicas entre los pacientes con y sin diabetes mellitus tipo 2 (DM2) bajo tratamiento de hemodiálisis (HD). Materiales y métodos: Estudio observacional de cohorte retrospectiva de pacientes atendidos por el Programa de Salud renal en el Centro de Prevención de Enfermedad Renal S.A.C (CENPER) de Lima, Perú. Se compararon los parámetros hematológicos y bioquímicos de 3 pacientes con DM2 y 3 pacientes sin DM2 sometidos a HD. Resultados: Se encontraron diferencias significativas (p<0,05) en el porcentaje de linfocitos, el cociente linfocito/ monocito (LMR), la concentración de hemoglobina y hematocrito (menor en pacientes diabéticos), en el porcentaje de monocitos y en el cociente neutrófilo/ linfocitos (NLR) (mayor en pacientes diabéticos). En los parámetros bioquímicos solo se encontró diferencia significativa en la transaminasa TGO que está más elevada en pacientes diabéticos comparados con pacientes no diabéticos (p<0.005). Conclusiones: Diabetes es un factor importante asociado con inflamación, anemia, linfopenia y monocitosis en pacientes sometidos a HD. LMR fue el marcador más potente de inflamación en esta serie de pacientes. Estudios a mayor escala son requeridos para corroborar esta evidencia.

Objective: To report if there are hematologic and biochemical differences among patients with and without type 2 diabetes mellitus (T2DM) undergoing hemodialysis (HD). Materials and methods: An observational, retrospective, cohort study of patients treated in the Renal Health Program at the Centro de Prevención de Enfermedad Renal S.A.C. (CENPER) in Lima, Peru. The hematologic and biochemical parameters of 3 patients with T2DM and 3 patients without T2DM undergoing HD were compared. Results: Significant differences (p < 0.05) were found in the lymphocyte percentage, lymphocyte/monocyte ratio (LMR), hemoglobin and hematocrit concentration (lower in diabetic patients), monocyte percentage, and neutrophil/ lymphocyte ratio (NLR) (higher in diabetic patients). In the biochemical parameters, the only significant difference was found in the glutamic-oxaloacetic transaminase (GOT) value, which was higher in the diabetic patients compared with the non-diabetic patients (p < 0.005). Conclusions: Diabetes is an important factor linked to inflammation, anemia, lymphopenia and monocytosis in patients undergoing HD. The LMR was the most powerful marker of inflammation in this patient series. Larger-scale studies are required to verify this evidence.

Cytokine mRNA in BALB/c mouse corneas infected with herpes simplex virus.

PURPOSE: To investigate cytokine mRNA expression and the influence of acyclovir and tetrandrine on that expression in the corneas of mice infected with herpes simplex virus type 1 (HSV-1). METHODS: Male BALB/c mice were infected in the right cornea with HSV-1. The corneas were harvested from control normal mice and from untreated, acyclovir-treated and tetrandrine-treated mice 14 days after infection. The infected corneas of each group were divided into inflamed and uninflamed depending on clinical observation. After total mRNA extraction from the corneas, gene expression of interleukin 1 beta (IL-1 beta), IL-6, tumour necrosis factor alpha (TNF-alpha) and transforming growth factor (beta (TGF-beta) was analysed by reverse transcription polymerase chain reaction. RESULTS: No mRNA expression of the cytokines was found in normal corneas. IL-1 beta and TNF-alpha mRNA was seen in inflamed corneas, while mRNA expression of IL-6 and relatively weaker TGF-beta mRNA expression were found both in inflamed corneas and in infected but uninflamed corneas treated with acyclovir. TNF-alpha mRNA was present in the uninflamed corneas of tetrandrine-treated mice. No influence of either agent was found on TGF-beta gene expression. CONCLUSIONS: The results suggest that local IL-1 beta and TNF-alpha gene expression is required for corneal inflammation, whereas IL-6 and TGF-beta may exert antiviral and inflammation regulatory activities in HSV corneal infection.

The role of cytokines in experimental herpes simplex keratitis.

Experimental corneal infection with herpes simplex virus 1 (HSV-1) resulted in 11-21 days in herpes simplex keratitis (HSK) in C.A1-20 but not C.B-17 strain of BALB/c Igh-1-disparate mice. Formation of mRNAs of various pro-inflammatory cytokines was analyzed in corneas and draining lymph nodes (LNs) of HSK-susceptible C.A1-20 and HSK-resistant C.B-17 mice following HSV-1 corneal inoculation by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. Transcripts for interleukin (IL)-2 and interferon (IFN)-gamma were expressed in LNs of susceptible but not resistant mice. The level of IL-6 expression in the cornea correlated with the severity of keratitis in susceptible mice, being evident at days 4 and 14 after virus inoculation and thus showing a biphasic response. Resistant mice did not develop HSK and did not express IL-6. The IL-1beta and IL-4 gene transcription began early (day 7) in the corneas of resistant mice and then ceased, while in the corneas of susceptible mice, it began later (day 11). Taken together, these results indicate that IL-1beta, IL-4, IL-6, and IL-7 participate in the local inflammatory response in HSK.

Analysis of T cell receptor V beta gene expression in B cell deficient mice after experimental herpes simplex virus keratitis.

To examine the importance of B cells in the regulation of the T cell response to herpes simplex virus (HSV) infection, we have analyzed the selection of the T cell receptor (TCR) repertoire in C.B-17 mice that lack B cells (B. mice) compared with age-matched immunocompetent C.B-17 mice, usually resistant to herpes simplex keratitis (HSK). TCR V beta transcripts used by these mice were analyzed by polymerase chain reaction (PCR) with variable gene-specific primers. Clinical examination showed that the incidence of HSK was significantly different between untreated (control) and anti-mu antibody (Ab)-treated mice (p < 0.0001). Passive transfer of anti-HSV Ab into B. mice, before infection, prevented HSK; transfer of naive B cells allowed HSK to evolve in 50% of these mice. AT the level of gene expression, we demonstrated that the anti-mu Ab treatment altered TCR V beta gene expression in eyes, spleen, thymus and lymph nodes (LN) of C.B-17 mice. Preferential utilization of a single TCR Tb gene was not detected in the course of the disease except in LN, although in resistant mice there were different patterns of mRNA induction in T cells expressing specific TCR Vb elements that were not seen in susceptible mice, namely the lack of expression of V beta 8.1, V beta 8.2 and V beta 8.3 in eyes, the expression of V beta 7 in spleen, and the lack expression of V beta 6 and V beta 13 in thymus. These observations together with previous findings suggest that at the level of protein production, anti-HSV Ab not only can provide protection against HSK but is also a critical component for protection against HSV in normally resistant C.B17 mice, and that a dysregulation of the immune system in B. mice is manifested by dramatic changes in TCR V beta usage at the molecular level.

Expression of collagens I, III, IV and V mRNA in excimer wounded rat cornea: analysis by semi-quantitative PCR.

A semi-quantitative polymerase chain reaction (PCR) methodology was used to evaluate the kinetic changes occurring in collagens I, III, IV and V mRNA in rat cornea following excimer laser keratectomy. cDNA was synthesized from RNA extracted from rat cornea at various times following excimer laser photoablative keratectomy. Collagen cDNA sequences were subsequently amplified using specific sets of oligonucleotide primers. Competitive PCR amplification was carried out using an internal standard so that a semi-quantitative analysis of message for synthesis of collagen types I, III, IV and V could be performed and time course dynamics of message for these collagens studied. There was a biphasic increase in the levels of collagens III, IV and V mRNA following excimer laser keratectomy. Collagen I mRNA levels demonstrated a more sustained increase and were still elevated at 6 weeks following wounding. Collagens IV and V mRNA showed the largest increase with an approximate three fold increase over controls between 4 days and 1 week. Our results demonstrate that upregulation of stromal collagens I, III, and V mRNA and basement membrane collagen IV mRNA occurs in rat cornea following excimer laser keratectomy.

Variable region usage in B cell deficient mice in a model of experimental herpes simplex virus retinitis.

By using PCR, we have found previously that differences in T cell receptor alpha/beta chain variable region (TCR alpha/beta) gene expression in lymph nodes (LN) existed between susceptible and resistant mice following anterior chamber (AC) inoculation with herpes simplex virus (HSV). We now report and compare the immunoglobulin gene variable region (Ig VH) and the TCR V beta mRNA expression in normal congenic mice (BALB/c and C.B-17) and also in B cell modulated C.B-17 mice (B-). RNA prepared from spleen and LN of these mice before and after HSV-AC inoculation was analysed by PCR using oligoprimers specific for 11 VH gene families and 19 V beta gene families. Densitometry analysis revealed that VH family mRNA expression levels in spleen and lymph nodes did not correlate with HSV susceptibility or resistance patterns in B-, BALB/c and C.B-17 mice. Analysis of TCR V beta chain genes showed that spleen of resistant C.B-17 and B-Ab transferred mice showed a preference for V beta 11 gene expression not seen in susceptible mice. In contrast, LN of BALB/c and B-, both susceptible mice, made TCR V beta transcripts that were indistinguishable from those generated by resistant C.B-17 mice, following HSV-AC inoculation. Finally, TCR V beta gene family repertoire appears to be severely diminished in uninfected B- mice (50% in LN and 45% in spleen) by anti-mu treatment.

T cell receptor V beta gene expression in experimental herpes stromal keratitis.

Our study examined T cell receptor (TCR) V beta mRNA expression in a murine model of experimental herpes simplex keratitis (HSK). We employed a polymerase chain reaction (PCR) technique to detect TCR V beta mRNA expression in the inoculated eyes of both HSK-susceptible and HSK-resistant mice at different time points after corneal inoculation with herpes simplex virus type 1 (HSV-1), followed by Southern blotting and densitometry analysis. In eyes from HSK-susceptible C.AL-20 mice, a more diverse TCR V beta transcript usage pattern was detected as compared with that seen in HSK-resistant C.B-17 mice. V beta 8 family members were expressed in both strains of mice at days 11, 14 and 21 post-inoculation. By densitometry, at day 11, the intensity of expression of V beta 8.2 and V beta 8.3 message was significantly greater in the eyes of C.AL-20 mice; V beta 8.1 was expressed only in C.B-17 mice. There were obvious differences in the TCR V beta expression between HSK-susceptible and HSK-resistant mice. The differences in the intensity of the message expressed by V beta 8 family members between the two strains could be correlated to previous experiments that showed V beta 8.1,2+ T cells as the main infiltrating cells in the corneas of HSK-susceptible mice by day 11 and 14 after challenge with HSV-1.

The role of B lymphocytes in experimental herpes simplex viral retinitis.

The purpose of this study was to examine B cell participation in experimental herpes simplex virus (HSV) retinitis. Passive immunization with anti-herpes antibody protects BALB/c mice from herpes simplex retinitis (HSR). Using anti-Mu antibody treatment, we modified the B cell population of C.B-17 mice, normally resistant to HSR, in order to test the hypothesis that such treatment would render them susceptible to HSR by impairing their early antibody response to anterior chamber (AC) inoculation with HSV. We analysed the effect of anti-Mu treatment on their susceptibility to HSR and then employed Polymerase Chain Reaction (PCR) and ELISA techniques to study the patterns of immunoglobulin gene and protein expression, and the T-cell receptor alpha/beta (TCR alpha/beta) gene expression after AC inoculation of HSV. Immunohistopathologic analysis revealed that 100% of the B cell deficient mice (B-) developed contralateral retinitis following AC inoculation, confirming the hypothesis that anti-Mu antibody treatment would convert HSR-resistant mice into HSR-susceptible ones. Transfer of B cells from naive congenic donor mice resulted in 67% of recipient B- mice developing contralateral retinitis. Transfer of anti-HSV antibody conferred nearly complete protection, with only 11% of mice developing retinitis (P < 0.005). PCR and ELISA analysis showed that both untreated and B- C.B-17 mice showed similar dynamic patterns of mRNA IgG isotype expression and of anti-HSV IgG isotypic antibody response following AC inoculation. Thus, we were forced to reject the hypothesis that an impaired early antibody response is primarily responsible for the increased HSR susceptibility seen in B- mice. In contrast, PCR analysis of TCR alpha/beta mRNA expression revealed dramatic differences between susceptible and resistant mice, suggesting that TCR V beta selection and usage may be a critical factor influencing HSR-sensitivity in this murine model, and that B cells (and immunoglobulin isotype) may play a role in TCR V beta selection and usage after ocular encounter with HSV.

Differential expression of alternatively spliced fibronectin in normal and wounded rat corneal stroma versus epithelium.

PURPOSE: The polymerase chain reaction was used to examine fibronectin (FN) expression during corneal scrape wounding with specific attention to the presence, absence, or gross changes of alternatively spliced FN as differentially expressed in the corneal stroma versus the epithelium in normal and wounded tissue. METHODS: Specific FN cDNA sequences were synthesized from rat cornea with total RNA and were amplified using various sets of synthetic oligonucleotide primers. RESULTS: The authors observed the presence and sustained the expression of total FN, EIIIA, EIIIB, and V-region FN mRNA in normal and injured corneal stroma for up to 3 weeks after scrape wounding. In contrast, complementary overlying epithelial samples were virtually devoid of FN message. CONCLUSIONS: These data suggest that functionally different, alternatively spliced FN isoforms may be involved both in the maintenance of the normal cornea and in wound healing, and that their synthesis occurs in situ principally by the stroma rather than by the epithelium.

Expression of fibronectin isoforms in rat cornea after an epithelial-scrape wound.

The polymerase chain reaction (PCR) technique was used to analyze the presence or absence of the EIIIB and V region fibronectin (FN) mRNA isoforms, generated by alternative splicing of the FN primary mRNA transcript during epithelial wound healing, in a rat cornea wound model. A central 4mm area of the cornea was denuded of epithelium. At 5, 15, 30, 45 minutes, 1, 2, 4, 8, 24, 48 hours, and 4 days post-wounding, the rats were euthanized and a 6mm diameter full thickness corneal biopsy was performed. cDNA was synthesized from extracted RNA, and specific FN sequences were amplified by PCR as directed by different sets of 5' and 3' primers specific for the alternatively spliced EIIIB and V region domains. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used to normalize the data. PCR products were analyzed by agarose gel electrophoresis and the identity of the bands were confirmed by direct nucleotide sequencing. The alternatively-spliced EIIIB and all three alternatively spliced isoforms of V domain FN mRNA were observed post-wounding. Similarly, in normal tissue, EIIIB and all three V region alternatively spliced FN isoforms were expressed. These findings suggest that in situ synthesis of cellular associated EIIIB FN and FN V domain may play a role in corneal wound healing.